This Article Abstract Full Text (PDF) Data Supplement All Versions of this Article: 23/3/461    most recent 01.ATV.0000060462.35946.B3v1 Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Rozenberg, O. Articles by Aviram, M. Search for Related Content PubMed PubMed Citation Articles by Rozenberg, O. Articles by Aviram, M. Related Collections Pathophysiology Genetically altered mice Lipid and lipoprotein metabolism

(Arteriosclerosis, Thrombosis, and Vascular Biology. 2003;23:461.)
© 2003 American Heart Association, Inc.

Atherosclerosis and Lipoproteins

Human Serum Paraoxonase 1 Decreases Macrophage Cholesterol Biosynthesis

Possible Role for Its Phospholipase-A₂–Like Activity and Lysophosphatidylcholine Formation

Orit Rozenberg; Diana M. Shih; Michael Aviram

From the Lipid Research Laboratory (O.R., M.A.), Technion Faculty of Medicine, The Rappaport Institute for Research in Medical Sciences, Rambam Medical Center, Haifa, Israel; and the Department of Medicine (D.M.S.), UCLA, Los Angeles, Calif.

Correspondence to Prof. Michael Aviram, The Lipid Research Laboratory, Rambam Medical Center, Haifa, Israel, 31096. E-mail aviram{at}tx.technion.ac.il

	Abstract

Top Abstract Introduction Methods Results Discussion References

 
Objective— Human serum paraoxonase 1 (PON1) activity is inversely related to the risk of developing an atherosclerotic lesion, which contains cholesterol-loaded macrophage foam cells. To assess a possible mechanism for this relationship, we analyzed the effect of PON1 on cellular cholesterol biosynthesis.

Methods and Results— Mouse peritoneal macrophages (MPMs) were harvested from PON1-deficient mice (PON1° and PON1°/E° mice on the genetic background of C57BL/6J and E° mice, respectively). PON1°/E° mice exhibited a significantly 51% increased atherosclerotic lesion area and 35% increased macrophage cholesterol content compared with control E° mice. In parallel, macrophage cholesterol biosynthesis rates were increased in PON1-deficient mice MPMs by 50% compared with their controls. Incubation of macrophages with human PON1 revealed a dose-dependent inhibitory effect (up to 84%) on macrophage cholesterol biosynthesis. We demonstrated a PON1 phospholipase-A₂–like activity on MPMs, evidenced by release of polyunsaturated fatty acids and formation of lysophosphatidylcholine. On incubation of macrophages with lysophosphatidylcholine, a dose-dependent inhibition (up to 40%) of cellular cholesterol biosynthesis was noted. The inhibitory effect of PON1 on macrophage cholesterol biosynthesis was shown to be downstream to mevalonate, probably at the lanosterol metabolic point.

Conclusions— PON1 inhibits macrophage cholesterol biosynthesis and atherogenesis probably through its phospholipase-A₂–like activity.

Key Words: paraoxonase • macrophage • cholesterol biosynthesis • phospholipase-A₂ • lysophosphatidylcholine

	Introduction

Top Abstract Introduction Methods Results Discussion References

 
Macrophage cholesterol accumulation and foam cell formation are the hallmark of early atherogenesis.^1,2 Cellular cholesterol accumulation can result from increased uptake of LDL or its modified forms,^3,4 from decreased cellular cholesterol efflux,⁵ and/or from enhanced cellular cholesterol biosynthesis.⁶ In the atherosclerotic lesion, which is characterized by the accumulation of cholesterol-loaded macrophages, the presence of paraoxonase 1 (PON1) was shown.^7,8 Human serum paraoxonase is an HDL-associated esterase, which possess antiatherosclerotic properties.^7,9–11 Human PON1 transgenic mice reveal decreased atherosclerotic lesions compared with control mice.⁹ Moreover, in atherosclerotic patients decreased serum PON1 activities have been shown,^12,13 and mice fed an atherogenic diet exhibit decreased PON1 mRNA levels.¹⁴ PON1-deficient mice fed a high-fat diet demonstrate accelerated atherosclerosis compared with control mice.^15,16 PON1 possesses multiple hydrolyzing activities toward organophosphates,^17,18 lipid peroxides,^7,19,20 and lactones.²¹ PON1 can also hydrolyze proteoliposome–phosphatidylcholine-core aldehydes and phosphatidylcholine–isoprostanes at position sn-2 to yield the formation of lysophosphatidylcholine (LPC) by a phospholipase-A₂ (PLase-A₂)–like activity.^22,23 The presence of a hydrolase, such as PON1, in arterial lesion areas^7,8 may have a protective effect against cellular cholesterol accumulation, which can possibly explain the inverse relationship between serum PON1 activity and atherosclerosis.^9–12 Thus, we investigated cellular cholesterol biosynthesis in peritoneal macrophages harvested from PON1-deficient mice as well as the direct effect of PON1 on macrophage cholesterol biosynthesis.

	Methods

Top Abstract Introduction Methods Results Discussion References

 
Mice
PON1-deficient mice (PON1° and PON1°/E° mice) were obtained as previously described.¹⁵ C57BL/6J and E° mice were used as controls. Two-month-old male mice were all fed regular chow diet.

PON1 Activity Measurement
Serum PON1 activity toward phenylacetate (arylesterase activity) was measured spectrophotometrically at 270 nm.¹⁷ For the determination of PON1 arylesterase activity in cells, phenylacetate in the assay mixture was added to the washed cells, and after 5 minutes incubation at room temperature, the supernatant was measured spectrophotometrically at 270 nm.

Purification of PON1 from Human Serum
PON1 was purified from the sera of healthy human volunteers identified as homozygous for PON1Q as described previously.⁷ Before addition to the cells, tergitol residues were removed by Extracti-Gel D Detergent Removing Gel (10 mL, Pierce, Ill).

Lipoprotein Separation
Serum HDL, isolated from fasted normolipidemic volunteers, and mouse HDL, derived from PON1° and C57BL/6J mice, were prepared by discontinuous density gradient ultracentrifugation.¹¹ HDLs (1.210 g/mL) were dialyzed against 150 mmol/L NaCl and 1 mmol/L CaCl₂ (pH 7.4, 4°C).

Histopathology of Aortic Atherosclerotic Lesion
PON1-deficient mice and their controls were euthanized and their aortas were dissected and fixed in 3% glutaraldehyde in 0.1 mol/L sodium cacodylate buffer, pH 7.4, followed by postfixation for 4 hours in 1% osmium tetroxide (in water). The samples were stored in 0.1 mol/L sodium cacodylate buffer containing 7.5% wt/vol sucrose before treatment with an unbuffered 1% aqueous solution of osmium tetroxide for 4 hours. The prolonged osmium treatment stains the intraluminal, intramural, and intracellular lipids a dense black color. The lesion areas were determined by using a computerized quantitative image-analysis system (Olympus Cue-2, Lake Success, NY) with morphometric software.²⁴

Cells
J774A.1 murine macrophage cells and Chinese hamster ovary (CHO) cells were purchased from the American Tissue Culture Collection (ATCC, Rockville, Md). Cultures of bovine corneal endothelial cells were established from steer eyes as described elsewhere.²⁵

Mouse peritoneal macrophages (MPMs) were harvested from peritoneum fluid 4 days after intraperitoneal injection of 3 mL of thioglycolate (40 g/L saline).⁶ The cells (10 to 20x10⁶/mouse) were resuspended in DMEM (with 100,000U/L penicillin, 100 mg/L streptomycin, and 2 mmol/L of glutamine) containing 5% FCS.

Macrophage Cholesterol Content
Cellular protein content was determined by using the method of Lowry et al.²⁶ MPMs (3x10⁶/well) sonicated in PBS containing 10 µmol/L butylated hydroxytoluene were extracted with 3 volumes of diethyl ether containing 5-cholestane (10 µL of 50 µg/mL stock) as internal standard. The upper phase was dried and cholesterol content in the samples was measured, after saponification, by gas chromatography-mass spectrometry (GC-MS) analysis.²⁷

Macrophage LPC Content
MPM (3x10⁶/well) were sonicated and extracted for cellular phospholipids with chloroform:methanol (2:1, v:v), then the upper phase was removed and the lower phase was dried. The samples were dissolved in water:acetonitrile (1:1, v:v). After addition of 5 mmol/L NaCl, LPC content in the samples was measured by liquid chromatography-mass spectrometry analysis.²⁸

Fatty Acid Release from Macrophages
Arachidonic Acid
The medium from MPMs (2x10⁶/well) was collected and extracted with 4 mL of diethyl ether (with 10 µmol/L butylated hydroxytoluene). The residue of the upper phase dissolved in methanol was injected to high-performance liquid chromatography (HPLC).²⁹

[¹⁴C]-Linoleic Acid
MPMs (2x10⁶/well) were incubated at 37°C for 2 hours in DMEM with 1-palmitoyl-2-[¹⁴C]-linoleyl-phosphatidylcholine (0.125 µCi/mL, Amersham Biosciences, Buckinghamshire, UK), followed by cell washes with PBS and a further incubation with purified PON1 for up to 3 hours. Samples were extracted with diethyl ether and run on thin-layer chromatography (TLC) and developed in hexane:ether:acetic acid (130:30:1.5, v:v:v). Free [¹⁴C]-linoleic acid spots were visualized by iodine vapor (by using standard for identification), and counted in a ß-counter.

Macrophage Cholesterol Biosynthesis
Cellular cholesterol biosynthesis was assayed after incubation of macrophages (2x10⁶/well) with increasing concentrations of purified PON1, HDL, or LPC for 18 hours at 37°C with DMEM containing 2% BSA followed by additional 3 hours of incubation at 37°C with [³H]-acetate (3.3 µCi/mL) or [³H]-mevalonate (2.3 µCi/mL). Cellular lipids were extracted with hexane:isopropanol (3:2, v:v), and the upper phase was dried under nitrogen. The lipids were then separated by TLC and developed in hexane:ether:acetic acid (130:30:1.5, v:v:v). Unesterified cholesterol, lanosterol, and linoleic acid spots were visualized by iodine vapor (by using standard for identification) and counted with ß-counter.⁶ Lanosterol spots were further analyzed by GC-MS, but no contamination of cholesterol or other precursors of cholesterol could be detected in the lanosterol spot.

PON1/2/3 mRNA Expression by Reverse Transcriptase Polymerase Chain Reaction (RT-PCR)
Total RNA was extracted from MPMs with Tri-reagent. cDNA were generated from 1 µg of total RNA by using RT. Products of the RT reaction were subjected to PCR amplification by using the following primers. For PON1, forward primer: 5'-TAGAGATTCTGCCTAA-TGGAC-3', reverse primer: 5'-GCAGGACGGTACCATTCTC-3'; for PON2: 5'-GATGGATCTGGACGAGAGAC-3', reverse primer: 5'-GAATCAAACCCTTCTGCCAC-3'; and for PON3: 5'-CACTG-CTTATCTTTATGTCGTG-3', reverse primer: 5'-GAAGCACAG-AGCCGTTGTTC-3'. Similar conditions were used to amplify glyceraldehydes-3-Phosphate-dehydrogenase (GAPDH), as previously described.⁶

The PCR program used for the PONs was as follows: 1 cycle (1 minute at 94°C), 30 cycles for PON3 and GAPDH, or 40 cycles for PON1 and PON2 (30 seconds at 94°C, 30 seconds at 57°C, 1 minute at 68°C), 1 cycle (7 minutes at 68°C). The cDNA products were separated on 1% agarose gel containing ethidium bromide.

Site-Directed Mutagenesis, Transfection, and Expression of Recombinants
Recombinants of PON1 (wild-type PON1Q and mutants with alanine or serine in the place of cystein-284) were produced as described previously.^7,19,30 MPM from PON1° mice were incubated with Ultra Culture (Cambrex Bioscience) media containing various PON1 preparations (1.3 arylesterase U/mL).

Statistical Analysis
Student’s t test was performed for all statistical analyses. For statistical analysis of parameters without Gaussian distributions, such as atherosclerotic lesions size, the nonparametric Wilcoxon rank sum test was used. All results represent mean±SD.

	Results

Top Abstract Introduction Methods Results Discussion References

 
Atherosclerotic Lesion and Macrophage Cholesterol Content in PON1-Deficient Mice
Two populations of PON1 knockout (KO) mice were studied: PON1-KO/C57BL/6J (PON1°) mice and PON1-KO/apoE-KO (PON1°/E°) mice. These mice were compared with control groups with the appropriate genetic background, that is, the C57BL/6J and the E° mice, respectively. Whereas no significant atherosclerotic lesions were found in PON1° mice or in C57BL/6J mice at 12 months of age under a chow diet, the atherosclerotic lesions of PON1°/E° mice at 12 months of age under chow diet were more advanced and complicated than control E° mice atherosclerotic lesions. This was evidenced by increased content of cholesterol crystals (marked as I, found in Figure 1A and 1B), and by appearance of lipid droplets (marked as II, found in Figure 1B). In addition, in PON1-deficient mice, the lesion size was significantly larger (P<0.01) by 51%, compared with the lesion size in the control mice (Figure 1C).

View larger version (72K): [in this window] [in a new window]   Figure 1. Atherosclerotic lesion morphology and size and macrophage cholesterol content in PON1°/E° and in E° mice. The aortic arches derived from (A) E° mice and from (B) PON1°/E° were analyzed by using morphometric analyses. I, Cholesterol crystals (white needle-like aggregates). II, Lipids pools (dark area). C, Aortic arches lesion size. Results are presented as mean±SEM, (n=10). D, MPM total cholesterol content was determined by GC-MS analysis (n=6; *P<0.01; PON1°/E° vs E° mice).

Because cholesterol accumulation and macrophage foam cell formation are the hallmark of early atherogenesis and because MPMs resemble arterial macrophages, we analyzed the cholesterol content in MPMs harvested from these atherosclerotic mice. A significant (P<0.01) 35% increase in macrophage cholesterol content was found in MPM from PON1°/E° mice compared with MPM from E° mice (Figure 1D).

Macrophage Cholesterol Biosynthesis in MPMs Harvested from PON1-Deficient Mice
Because macrophage cholesterol content was increased in PON1-KO mice, we questioned whether PON1 deficiency was associated with enhanced macrophage cholesterol biosynthesis. In PON1° mice MPMs, cellular cholesterol biosynthesis was significantly increased by 50% (P<0.01) compared with cholesterol biosynthesis in control C57BL/6J mice MPMs (Figure 2A). Similarly, cholesterol biosynthesis in PON1°/E° mice MPMs was significantly increased by 51% (P<0.01) compared with cholesterol biosynthesis in control E° mice MPMs (Figure 2B).

View larger version (15K): [in this window] [in a new window]   Figure 2. Macrophage cholesterol biosynthesis in MPMs harvested from PON1-deficient mice compared with their appropriate control groups. Cholesterol biosynthesis in MPM from PON1° mice (A), from PON1°/E° mice (B), and from the control mice (A and B) was determined by TLC by using 3[H]-acetate as the precursor. (n=6; *P<0.01; PON1° vs C57BL/6J and PON1°/E° vs E° mice).

Like PON1, PON2 and PON3 are members of the PON gene family and are located adjacent to each other on chromosome 6 in mice.³¹ Thus, we questioned whether PON1 deficiency was associated with concomitant macrophage deficiency of PON2 and PON3. We found unchanged macrophage PON2 and PON3 (but not PON1) mRNA expression in PON1° MPMs by RT-PCR (data not shown), indicating that despite the absence of PON1, PON2 and PON3 are present in MPM from PON1° mice.

Direct Effect of Pure PON1 and HDL on Macrophage Cholesterol Biosynthesis
To study whether PON1 directly decreased macrophage cholesterol biosynthesis, we first incubated human PON1 (7.5 arylesterase U/mL) with PON1° mice MPMs. We observed a 40% increase in PON1 activity (toward phenylacetate) in treated cells compared with cells that were not incubated with PON1 (70 mU/mg cell protein versus 49 mU/mg cell protein, respectively), indicating PON1 association with macrophages. On incubation of PON1° mice MPMs with increasing concentrations of purified human PON1, a dose-dependent decrease in macrophage cholesterol biosynthesis rates by up to 64% (by using 7.5 arylesterase U/mL of PON1) was noted (Figure 3A). Because HDL is the physiological carrier of PON1 in serum, we examined a possible similar inhibitory effect of HDL on macrophage cholesterol biosynthesis. For this purpose we incubated MPMs from PON1° mice with human HDL. An HDL dose-dependent decrease in macrophage cholesterol biosynthesis by 21% and by 35% was noted by using 0.2 and 0.3 HDL arylesterase U/mL, respectively (475 and 715 µg HDL protein/mL; Figure 3B). Moreover, we found that HDL harvested from PON1° mice exhibited 91% decreased PON1 activity toward phenylacetate (arylesterase) compared with HDL harvested from C57BL/6J mice (1.1±0.1 U/mg HDL protein in C57BL/6J mice versus 0.1±0.01 U/mg HDL protein in PON1° mice). Thus, we examined the effect of the absence of PON1 in HDL harvested from PON1° mice on macrophage cholesterol biosynthesis. We found that incubation of PON1° mice MPMs with HDL harvested from PON1° mice revealed a 2.8-fold increment in macrophage cholesterol biosynthesis compared with cholesterol biosynthesis established after MPM incubation with HDL harvested from C57BL/6J mice (2123±113 versus 765±90 cpm/mg cell protein, respectively). To verify whether the inhibitory effect of PON1 on macrophage cholesterol biosynthesis was specific, several types of macrophages were incubated with increasing concentrations of PON1. Incubation of PON1°/E° mice MPM with increasing concentrations of PON1 revealed a dose-dependent decrease in macrophage cholesterol biosynthesis rates by up to 84% by using 7.5 arylesterase U/mL of PON1 (from 16400±395 to 6888±607 cpm/mg cell protein). Similarly, after incubation of MPM from C57BL/6J mice or of J774A.1 macrophage-like cell-line with PON1 (7.5 arylesterase U/mL), macrophage cholesterol biosynthesis rates decreased significantly (P<0.01) by 40% (from 10000±433 to 6016±500 cpm/mg cell protein) or by 62% (from 9837±260 to 3757±299 cpm/mg cell protein), respectively, compared with the cholesterol biosynthesis rates in cells that were incubated without PON1. These results indicate that PON1 specifically inhibits cholesterol biosynthesis in various types of macrophages. To find whether the effect of PON1 is selective to macrophages, we also used endothelial cells and fibroblasts (CHO cells). Whereas in endothelial cells PON1 decreased cholesterol biosynthesis, by 35% (from 2521±226 to 1636±197 cpm/mg cell protein), no significant decrease was detected in CHO cells (from 469±18 to 478±42 cpm/mg cell protein).

View larger version (30K): [in this window] [in a new window]   Figure 3. The effect of PON1 on PON1° MPM cholesterol biosynthesis. MPMs from PON1° mice were incubated with (A) PON1 (0, 0.5, 5, and 7.5 arylesterase U/mL), with (B) HDL (0, 0.2, and 0.3 arylesterase U/mL), or (C) with PON1 recombinants wild type (WT) or with two recombinant mutants where the cysteine at position-284 was replaced with either serine (serine-284 PON1 mutant) or alanine (alanine-284 PON1 mutant; 0.2 arylesterase U/mL). Then cellular cholesterol biosynthesis was determined by TLC by using 3[H]-acetate as the precursor (n=5; *P<0.01 vs treatment without PON1).

On searching for PON1 active site for its inhibitory effect on macrophage cholesterol biosynthesis we next used recombinant and mutant PON1. A significant decrease of 34% (P<0.01) in macrophage cholesterol biosynthesis was noted after incubating MPM from PON1° mice with recombinant wild type of PON1 (0.2 arylesterase U/mL; Figure 3C). The involvement of the only free sulfhydryl group in PON1, at position cysteine-284, in the inhibitory effect of PON1 on macrophage cholesterol biosynthesis was examined by substitution of the cysteine-284 by serine or alanine by using site-directed mutagenesis. Both substitutions revealed a significant (P<0.01) decreased capability of the PON1 mutants (0.2 arylesterase U/mL) to inhibit PON1° MPM cholesterol biosynthesis (decrement of only 8% and 17%, respectively, Figure 3C).

PLase-A₂–Like Activity of PON1 on Macrophage Phospholipids
PON1 inhibitory effect on macrophage cholesterol biosynthesis may have resulted from its PLase-A₂–like activity. On incubation of -palmitoyl-ß-arachidonyl--phosphatidylcholine (PAPC, 0.4 mg/mL) with 7.5 arylesterase U/mL of PON1 for 3 hours, HPLC analysis revealed 61% increase in arachidonic acid release (Figure 4A) compared with release in the absence of PON1.

View larger version (25K): [in this window] [in a new window]   Figure 4. PLase-A2–like activity of PON1. A, PAPC was incubated without (control) or with PON1 (+PON1, 7.5 arylesterase U/mL) and after lipid extraction, arachidonic acid levels were determined by HPLC (n=4; *P<0.01 vs control). B, MPM from PON1° mice were incubated for 2 hours with 1-palmitoyl-2-[14C]-linoleyl-phosphatidylcholine (0.125 µCi/mL), followed by further incubation without or with PON1 for up to 3 hours. Samples were extracted with diethyl ether and run on TLC for free [14C]-linoleic acid determination. (n=5; *P<0.01 vs control). C, PAPC was incubated with either serum from C57BL/6J mice or serum from PON1° mice after lipid extraction. Arachidonic acid levels were determined by HPLC. (n=4; *P<0.01; PON1° vs C57BL/6J).

We next questioned whether PON1 also possessed PLase-A₂–like activity toward macrophage phospholipids by using MPMs from PON1° mice that were preincubated with radiolabeled -palmitoyl-ß-linoleyl--phosphatidylcholine. By using TLC analysis, a time-dependent increased release of [¹⁴C]-linoleic acid from the cells was observed after incubation with PON1 (7.5 arylesterase U/mL) with up to 137% increase obtained after 3 hours of incubation compared with cells that were incubated without PON1 (Figure 4B). Similarly, 18 hours incubation of PON1° mice MPM with PON1 (7.5 arylesterase U/mL) revealed a substantial 6.9-fold increase (from 2.8±0.1 µmol to 19.3±1.4 µmol) in cellular LPC levels. LPC levels were also increased (by 23%) in C57BL/6J mice MPMs compared with LPC levels in PON1° mice MPMs (3.6±0.2 versus 2.8±0.1 µmol, respectively). To question the effect of PON1 presence in serum on PLase-A₂ activity, we measured this activity in serum from PON1° mice compared with serum from C57BL/6J mice and observed a 16% significant (P<0.01) decrement in the serum PLase-A₂ activity on PAPC as assessed by HPLC analysis of arachidonic acid release (Figure 4C).

LPC as a Possible Mediator for PON1 Inhibitory Effect on Macrophage Cholesterol Biosynthesis
PON1 PLase-A₂–like activity on phospholipids results in LPC formation.^22,23 Thus, we next questioned whether LPC, like PON1, had an inhibitory effect on macrophages cholesterol biosynthesis. Incubation of PON1° mice MPMs with increasing concentrations of LPC revealed a dose-dependent significant decrement (P<0.01) in macrophage cholesterol biosynthesis by up to 40% by using 15 µg/mL of LPC (see online Figure IA, which can be accessed at http://atvb. ahajournals.org). Similarly, Incubation of control C57BL/6J mice MPM with increasing concentrations of LPC revealed significant (P<0.01) dose-dependent inhibition of macrophage cholesterol biosynthesis by up to 31% by using 15 µg/mL of LPC (Figure IB).

Location of PON1 Action Along the Cholesterol Biosynthesis Pathway
3-Hydroxy-3-Methylglutaryl-Coenzyme-A-Reductase (HMG-CoA-Reductase) is the rate-limiting enzyme in the cellular cholesterol biosynthetic pathway. We next studied whether PON1 inhibited cholesterol biosynthesis by inhibiting the activity of this enzyme. For this purpose [³H]-acetate or [³H]-mevalonate was used as precursors for macrophage cholesterol biosynthesis in PON1° mice MPMs. A similar decrease of 64% (Figure IIA, which can be accessed at http://atvb.ahajournals.org) and of 58% (Figure IIB) in cellular cholesterol biosynthesis was observed by using these precursors, respectively. These results suggest that PON1 does not inhibit the HMG-CoA-reductase enzyme and that the inhibition of macrophage cholesterol biosynthesis pathway is located downstream to mevalonate.

LPC was shown to decrease cholesterol biosynthesis in liver cells, by inhibiting the conversion of lanosterol, an upstream product to cholesterol.³² Because in the present study LPC is shown to inhibit macrophage cholesterol biosynthesis, we next analyzed whether the inhibitory effect of PON1 on the macrophage cholesterol biosynthesis could be associated with the accumulation of lanosterol. Incubation of MPMs from PON1° mice with 7.5 arylesterase U/mL of PON1 revealed a substantial 4.3-fold or 4.4-fold increase in macrophage radiolabeled [³H]-lanosterol levels by using [³H]-acetate (Figure IIIA, which can be accessed at http://atvb. ahajournals.org) or [³H]-mevalonate (Figure IIIB) as precursors for cholesterol biosynthesis, respectively. This lanosterol accumulation, however, did not affect macrophage viability (analyzed by cell number and medium LDH activity). These findings suggest that PON1 inhibits the cholesterol biosynthesis pathway downstream to mevalonate, at the lanosterol metabolic step.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
The present study demonstrates, for the first time, that PON1 inhibits macrophage cholesterol biosynthesis. This was supported by increased cellular cholesterol biosynthesis in MPMs harvested from PON1-deficient mice and by the direct inhibitory effect of PON1 on macrophage cholesterol biosynthesis. Inhibition of macrophage cholesterol biosynthesis by PON1 is probably related to its PLase-A₂–like activity, which results in LPC formation, because LPC inhibits cellular cholesterol biosynthesis.

The increase in cholesterol biosynthesis in PON1-deficient mice MPM is probably not caused by inflammatory mediators, as PON1 was shown to have a specific direct effect on macrophage cholesterol biosynthesis on various types of macrophages. Furthermore, this effect of PON1 was not limited to macrophages because endothelial cells were also affected, in contrast to CHO fibroblasts. Thus, PON1 may selectively inhibit cellular cholesterol biosynthesis only in certain cells, such as arterial cells. The maximal PON1 activity toward phenylacetate (7.5 arylesterase U/mL) used in our in vitro studies was about 15% of that found in human serum, but similar to levels in the arterial wall.³³ Peritoneal macrophages, which are widely used to study cholesterol and lipoprotein metabolism in relation to atherogenesis, resemble arterial macrophages in their characteristics.³⁴

HMG-CoA-reductase, which converts HMG-CoA to mevalonate, is the rate-limiting enzyme in the cholesterol biosynthesis pathway.³⁵ HMG-CoA-reductase inhibitors (statins), the most efficient hypocholesterolemic drugs, decrease serum cholesterol levels by inhibiting cholesterol synthesis in hepatocytes³⁶ and in macrophages.³⁷ We thus questioned whether PON1 inhibition of macrophage cholesterol biosynthesis was located at the HMG-CoA-reductase action on HMG-CoA. However, by using [³H]-mevalonate as cholesterol biosynthesis precursor, PON1 decreased macrophage cholesterol biosynthesis to the same extent as it did by using [³H]-acetate as precursor, indicating that HMG-CoA-reductase is not the target for the inhibitory effect of PON1. It has been previously shown that PON1 can hydrolyze oxidized phospholipids in oxidized lipoproteins,³⁸ and PLase-A₂–like activity of PON1 was previously shown on proteoliposome–phosphatidylcholine-core aldehydes and on phosphatidylcholine–isoprostanes.^22,23 We extended this observation to macrophage phospholipids. Furthermore, we showed that PON1° mice serum compared with C57BL/6J mice serum had decreased PLase-A₂–like activity as analyzed by its capacity to hydrolyze PAPC and to release arachidonic acid. This decrement in PLase-A₂–like activity was relatively low because PLase-A₂ activities other than PON1 are also present in serum.³⁹ Like PAPC, other specific phospholipids and oxidized phospholipids in arterial macrophage foam cells can be appropriate substrates for PON1.

PLase-A₂ activity of PON1 results in the formation of LPC, and LPC inhibits liver cells cholesterol biosynthesis.³² Similarly, LPC dose-dependent inhibition of cholesterol biosynthesis in MPMs was observed. LPC was shown to decrease cholesterol biosynthesis by inhibiting the conversion of lanosterol to cholesterol.³² In the present study, accumulation of lanosterol was shown also in macrophages after treatment with PON1. Because the methodology available may not be sufficient to exclude additional sterols in the lanosterol TLC spots, the exact step of PON1 inhibition of cholesterol synthesis should be further studied.

HDL, the carrier of PON1, demonstrated, like PON1, a similar inhibitory effect on macrophage cholesterol biosynthesis by using similar PON1 activities toward phenylacetate. This effect of HDL is caused by the presence of PON1 in HDL, as PON1-deficiency attenuated the HDL inhibition of macrophage cholesterol biosynthesis. These results thus suggest that the effect of purified PON1 on cellular cholesterol biosynthesis is probably of physiological significance.

HDL is thought to have important role in reverse cholesterol transport from various cells (including arterial wall cells) to the liver. In macrophages, a retroendocytosis of HDL was shown,⁴⁰ where receptor-bound HDL internalizes into the cells. It is possible that through such a mechanism, PON1 can interact physiologically with macrophage phospholipids, resulting in the inhibition of cellular cholesterol biosynthesis. Moreover, PON1 retains its hydrophobic N-terminal sequence, which enables its association with phospholipids,⁴¹ on lipoproteins and on cells. Incubation of macrophages with PON1 revealed an increase in the cellular PON1 activity (toward phenylacetate). This phenomenon is in accordance with findings indicating PON1 location in the external membrane of PON1-transfected hepatocytes.⁴² HDL oxidation abolishes the ability of HDL to stimulate cellular cholesterol efflux. In the presence of PON1, however, the formation of oxidized HDL is decreased and the ability of HDL to induce macrophage cholesterol efflux increases.¹¹ PON1 may thus play important role in reverse cholesterol transport. Finally, the increased macrophage cholesterol biosynthesis observed in MPMs from PON1°/E° mice at 12 months of age fed a chow diet (4% fat) was associated with increased cellular cholesterol content in MPMs from these mice and with large complex atherosclerotic lesion. Increased atherosclerotic lesion was also previously shown in PON1°/E° mice at 3 months of age fed a 6% fat diet compared with control littermates.¹⁶ Similarly, in angiotensin-II–treated mice, increased macrophage cholesterol synthesis was associated with accelerated atherosclerosis progression.⁶

We thus conclude that PON1 deficiency is associated with increased macrophage cholesterol biosynthesis, cellular cholesterol accumulation, and atherogenesis. PON1 directly decreases macrophage cholesterol biosynthesis by its action downstream to mevalonate. PON1 PLase-A₂-like activity leads to LPC formation, and LPC may activate some signal transduction pathway(s), which results in the inhibition of macrophage cholesterol biosynthesis. Because cholesterol accumulation and foam cell formation are the hallmark of early atherogenesis, paraoxonase inhibitory effect on macrophage cholesterol biosynthesis and on macrophage oxidative stress^43,44 may have important antiatherosclerotic implications.

Received November 20, 2002; accepted January 9, 2003.

	References

Top Abstract Introduction Methods Results Discussion References

 
1. Glass CK, Witztum JL. Atherosclerosis: the road ahead. Cell. 2001; 104: 503–516.[CrossRef][Medline] [Order article via Infotrieve]

2. Aviram M. Macrophage foam cell formation during early atherogenesis is determined by the balance between pro-oxidants and anti-oxidants in arterial cells and blood lipoproteins. Antiox Redox Signal. 1999; 1: 585–594.[Medline] [Order article via Infotrieve]

3. Kruth HS, Huang W, Ishii I, Zhang WY. Macrophage foam cell formation with native low density lipoprotein. J Biol Chem. 2002; 277: 34573–34580.[Abstract/Free Full Text]

4. Steinberg D, Parthasarathy S, Carew TE, Khoo JC, Witztum JL. Beyond cholesterol: modifications of LDL that increase its atherogenicity. N Engl J Med. 1989; 320: 915–924.[Medline] [Order article via Infotrieve]

5. Braun A, Trigatti BL, Post MJ, Sato K, Simons M, Edelberg JM, Rosenberg RD, Schrenzel M, Krieger M. Loss of SR-BI expression leads to the early onset of occlusive atherosclerotic coronary artery disease, spontaneous myocardial infarctions, severe cardiac dysfunction, and premature death in apolipoprotein E-deficient mice. Circ Res. 2002; 90: 270–276.[Abstract/Free Full Text]

6. Keidar S, Attias J, Heinrich R, Coleman R, Aviram M, Angiotensin-II atherogenicity in apolipoprotein E-deficient mice is associated with increased cellular cholesterol biosynthesis. Atherosclerosis. 1999; 146: 249–257.[CrossRef][Medline] [Order article via Infotrieve]

7. Aviram M, Hardak E, Vaya J, Mahmood S, Milo S, Hoffman A, Billicke S, Draganov D, Rosenblat M. Human serum PON1 Q and R selectively decrease lipid peroxides in human coronary and carotid atherosclerotic lesions: PON1 esterase and peroxidase-like activities. Circulation. 2000; 101: 2510–2517.[Abstract/Free Full Text]

8. Mackness B, Hunt R, Durrington PN, Mackness MI. Increased immunolocalization of paraoxonase, clusterin, and apolipoprotein A-I in the human artery wall with the progression of atherosclerosis. Arterioscler Thromb Vasc Biol. 1997; 17: 1233–1238.[Abstract/Free Full Text]

9. Tward A, Xia YR, Wang XP, Shi YS, Park C, Castellani LW, Lusis AJ, Shih DM. Decreased atherosclerotic lesion formation in human serum paraoxonase transgenic mice. Circulation. 2002; 106: 484–490.[Abstract/Free Full Text]

10. Aviram M. Does paraoxonase play a role in susceptibility to cardiovascular disease? Mol Med Today. 1999; 5: 381–386.[CrossRef][Medline] [Order article via Infotrieve]

11. Aviram M, Rosenblat M, Bisgaier CL, Newton RS, Primo-Parmo SL, La Du BN. Paraoxonase inhibits HDL oxidation and preserves its functions: a possible peroxidative role for paraoxonase. J Clin Invest. 1998; 101: 1581–1590.[Medline] [Order article via Infotrieve]

12. Durrington PN, Mackness B, Mackness MI. Paraoxonase and atherosclerosis. Arterioscler Thromb Vasc Biol. 2001; 21: 473–480.[Abstract/Free Full Text]

13. Ayub A, Mackness MI, Arrol S, Mackness B, Patel J, Durrington PN. Serum paraoxonase after myocardial infarction. Arterioscler Thromb Vasc Biol. 1999; 19: 330–335.[Abstract/Free Full Text]

14. Shih DM, Gu L, Hama S, Xia YR, Navab M, Fogelman AM, Lusis AJ. Genetic-dietary regulation of serum paraoxonase expression and its role in atherogenesis in a mouse model. J Clin Invest. 1996; 97: 1630–1639.[Medline] [Order article via Infotrieve]

15. 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.[CrossRef][Medline] [Order article via Infotrieve]

16. Shih DM, Xia YR, Wang XP, Miller E, Castellani LW, Subbanagounder G, Cheroutre H, Faull KF, Berliner JA, Witztum JL, Lusis AJ. Combined serum paraoxonase knockout/apolipoprotein E-knockout mice exhibit increased lipoprotein oxidation and atherosclerosis. J Biol Chem. 2000; 275: 17527–17535.[Abstract/Free Full Text]

17. Gan K, 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]

18. Costa LG, Li WF, Richter RJ, Shih DM, Lusis A, Furlong CE. The role of PON1 in the detoxication of organophosphates and its human polymorphism. Chem Biol Interact. 1999; 119–120: 429–438.

19. Aviram M, Billecke S, Sorenson R, Bisgaier C, Newton R, Rosenblat M, Erogul J, Hsu C, Dunlop C, La Du BN. Paraoxonase active site required for protection against LDL oxidation involves its free sulfhydryl group and is different from that required for its arylesterase/paraoxonase activities: selective action of human paraoxonase allozymes Q and R. Arterioscler Thromb Vasc Biol. 1998; 18: 1617–1624.[Abstract/Free Full Text]

20. Navab M, Berliner JA, Watson AD, Hama SY, Territo MC, Lusis AJ, Shih DM, Van Lenten BJ, Frank JS, Demer LL, Edwards PA, Fogelman AM. The yin and yang of oxidation in the development of the fatty streak. Arterioscler Thromb Vasc Biol. 1996; 16: 831–842.[Abstract/Free Full Text]

21. Billecke S, Draganov D, Counsell R, Stetson P, Watson C, Hsu C, La Du BN. Human serum PON1 isozymes Q and R hydrolyze lactones and cyclic carbonate esters. Drug Metab Dispos. 2000; 28: 1335–1342.[Abstract/Free Full Text]

22. Ahmed Z, Ravandi A, Maguire GF, Emili A, Draganov D, La Du BN, Kuksis A, Connelly PW. Apolipoprotein A-I promotes the formation of phosphatidylcholine core aldehydes that are hydrolyzed by paraoxonase (PON-1) during HDL oxidation with a peroxynitrite donor. J Biol Chem. 2001; 276: 24473–24481.[Abstract/Free Full Text]

23. Ahmed Z, Ravandi A, Maguire GF, Emili A, Draganov D, La Du BN, Kuksis A, Connelly PW. Multiple substrates for paraoxonase-1 during oxidation of phosphatidylcholine by peroxynitrite. Biochem Biophys Res Commun. 2002; 290: 391–396.[CrossRef][Medline] [Order article via Infotrieve]

24. Plump AS, Smith JD, Hayek T, Aalto-Setala K, Walsh A, Verstuyft JG, Rubin EM, Breslow JL. Severe hypercholesterolemia and atherosclerosis in apolipoprotein E-deficient mice created by homologous recombination in ES cells. Cell. 1992; 71: 343–353.[CrossRef][Medline] [Order article via Infotrieve]

25. Goldshmidt O, Zcharia E, Abramovich R, Metzger S, Aingorn H, Friedmann Y, Schirrmacher V, Mitrani E, Vlodavsky I. Cell surface expression and secretion of heparanase markedly promote tumor angiogenesis and metastasis. Proc Natl Acad Sci U S A. 2002; 99: 10031–10036.[Abstract/Free Full Text]

26. Lowry OH, Rosebrough NJ, Farr L, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951; 193: 265–275.[Free Full Text]

27. Kaplan M, Aviram M, Knopf C, Keidar S, Angiotensin-II reduces macrophage cholesterol efflux: a role for the AT-1 receptor but not for the ABC1 transporter. Biochem Biophys Res Commun. 2002; 290: 1529–1534.[CrossRef][Medline] [Order article via Infotrieve]

28. Vesterqvist O, Sargent CA, Grover GJ, Warrack BM, DiDonato GC, Ogletree ML. Characterization of rabbit myocardial phospholipase-A2 activity using endogenous phospholipid substrates. Anal Biochem. 1994; 217: 210–219.[CrossRef][Medline] [Order article via Infotrieve]

29. Rosenblat M, Aviram M. Oxysterol-induced activation of macrophage NADPH-oxidase enhances cell-mediated oxidation of LDL in the atherosclerotic apolipoprotein E deficient mouse: inhibitory role for vitamin E. Atherosclerosis. 2002; 160: 69–80.[CrossRef][Medline] [Order article via Infotrieve]

30. Aviram M, Rosenblat M, Billecke S, Erogul J, Sorenson R, Bisgaier CL, Newton RS, La Du B. Human serum PON1 is inactivated by Ox-LDL and preserved by antioxidants. Free Radic Biol Med. 1999; 26: 892–904.[CrossRef][Medline] [Order article via Infotrieve]

31. Primo-Parmo SL, Sorenson RC, Teiber J, La Du BN. The human serum paraoxonase/arylesterase gene (PON1) is one member of a multigene family. Genomics. 1996; 33: 498–507.[CrossRef][Medline] [Order article via Infotrieve]

32. Shin BA, Kim YR, Lee IS, Sung CK, Hong J, Sim CJ, Im KS, Jung JH. Lyso-PAF analogues and lysophosphatidylcholines from the marine sponge Spirastrella abata as inhibitors of cholesterol biosynthesis. J Natl Prod. 1999; 62: 1554–1557.

33. Mackness MI, Mackness B, Arrol S, Wood G, Bhatnagar D, Durrington PN. Presence of paraoxonase in human interstitial fluid. FEBS Lett. 1997; 416: 377–380.[CrossRef][Medline] [Order article via Infotrieve]

34. Keller-Weibel G, Yancey PG, Jerome WG, Walser T, Mason RP, Phillips MC, Rothblat GH. Crystallization of free cholesterol in model macrophage foam cells. Arterioscler Thromb Vasc Biol. 1999; 19: 1891–1898.[Abstract/Free Full Text]

35. Sato R, Goldstein JL, Brown MS. Replacement of serine-871 of hamster 3-hydroxy-3-methylglutaryl-CoA-reductase prevents phosphorylation by AMP-activated kinase and blocks inhibition of sterol synthesis induced by ATP depletion. Proc Natl Acad Sci U S A. 1993; 90: 9261–9265.[Abstract/Free Full Text]

36. McTaggart F, Buckett L, Davidson R, Holdgate G, McCormick A, Schneck D, Smith G, Warwick M. Preclinical and clinical pharmacology of Rosuvastatin, a new 3-hydroxy-3-methylglutaryl-CoA-reductase inhibitor. Am J Cardiol. 2001; 87: 28B–32B.[Medline] [Order article via Infotrieve]

37. Koh KK. Effects of statins on vascular wall: vasomotor function, inflammation, and plaque stability. Cardiovasc Res. 2000; 47: 648–657.[Abstract/Free Full Text]

38. Watson AD, Berliner JA, Hama SY, La Du BN, Faull KF, Fogelman AM, Navab M. Protective effect of HDL associated paraoxonase: inhibition of the biological activity of minimally Ox-LDL. J Clin Invest. 1995; 96: 2882–2891.

39. Petrovic N, Grove C, Langton PE, Misso NL, Thompson PJ. A simple assay for a human serum phospholipase-A2 that is associated with HDLs. J Lipid Res. 2001; 42: 1706–1713.[Abstract/Free Full Text]

40. Schmitz G, Robenek H, Lohmann U, Assmann G. Interaction of HDLs with cholesteryl ester-laden macrophages: biochemical and morphological characterization of cell surface receptor binding, endocytosis and resecretion of HDLs by macrophages. EMBO J. 1985; 4: 613–622.[Medline] [Order article via Infotrieve]

41. 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.[Abstract/Free Full Text]

42. Deakin S, Leviev I, Gomaraschi M, Calabresi L, Franceschini G, James RW. Enzymatically active paraoxonase-1 is located at the external membrane of producing cells and released by a high affinity, saturable, desorption mechanism. J Biol Chem. 2002; 277: 4301–4308.[Abstract/Free Full Text]

43. Rozenberg O, Rosenblat M, Coleman R, Shih DM, Aviram M. Paraoxonase (PON1) deficiency is associated with increased macrophage oxidative stress: studies in PON1-knockout mice. Free Radic Biol Med. In press.

44. Rosenblat M, Draganov D, Watson CE, Bisgaier CL, La Du BN, Aviram M. Mouse macrophage paraoxonase 2 activity is increased whereas cellular paraoxonase 3 activity is decreased under oxidative stress. Arterioscler Thromb Vasc Biol. 2003; 23: 468–474.[Abstract/Free Full Text]

This article has been cited by other articles:

				F. F. Verit, O. Erel, and N. Celik Serum paraoxonase-1 activity in women with endometriosis and its relationship with the stage of the disease Hum. Reprod., January 1, 2008; 23(1): 100 - 104. [Abstract] [Full Text] [PDF]

				L. Gaidukov and D. S. Tawfik The development of human sera tests for HDL-bound serum PON1 and its lipolactonase activity J. Lipid Res., July 1, 2007; 48(7): 1637 - 1646. [Abstract] [Full Text] [PDF]

				L. Gaidukov, M. Rosenblat, M. Aviram, and D. S. Tawfik The 192R/Q polymorphs of serum paraoxonase PON1 differ in HDL binding, lipolactonase stimulation, and cholesterol efflux J. Lipid Res., November 1, 2006; 47(11): 2492 - 2502. [Abstract] [Full Text] [PDF]

				M. Rosenblat, L. Gaidukov, O. Khersonsky, J. Vaya, R. Oren, D. S. Tawfik, and M. Aviram The Catalytic Histidine Dyad of High Density Lipoprotein-associated Serum Paraoxonase-1 (PON1) Is Essential for PON1-mediated Inhibition of Low Density Lipoprotein Oxidation and Stimulation of Macrophage Cholesterol Efflux J. Biol. Chem., March 17, 2006; 281(11): 7657 - 7665. [Abstract] [Full Text] [PDF]

				J. F. Teiber, D. I. Draganov, and B. N. La Du Purified human serum PON1 does not protect LDL against oxidation in the in vitro assays initiated with copper or AAPH J. Lipid Res., December 1, 2004; 45(12): 2260 - 2268. [Abstract] [Full Text] [PDF]

				M. Rosenblat, D. Draganov, C. E. Watson, C. L. Bisgaier, B. N. La Du, and M. Aviram Mouse Macrophage Paraoxonase 2 Activity Is Increased Whereas Cellular Paraoxonase 3 Activity Is Decreased Under Oxidative Stress Arterioscler. Thromb. Vasc. Biol., March 1, 2003; 23(3): 468 - 474. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Data Supplement All Versions of this Article: 23/3/461    most recent 01.ATV.0000060462.35946.B3v1 Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Rozenberg, O. Articles by Aviram, M. Search for Related Content PubMed PubMed Citation Articles by Rozenberg, O. Articles by Aviram, M. Related Collections Pathophysiology Genetically altered mice Lipid and lipoprotein metabolism