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(Circulation. 2005;111:2798-2804.)
© 2005 American Heart Association, Inc.

Vascular Medicine

Expression of Human Myeloperoxidase by Macrophages Promotes Atherosclerosis in Mice

Timothy S. McMillen, PhD; Jay W. Heinecke, MD; Renee C. LeBoeuf, PhD

From the Nutritional Sciences Interdisciplinary Program (T.S.M., R.C.L.) and the Department of Medicine (T.S.M., R.C.L., J.W.H.), University of Washington, Seattle.

Correspondence to Renée C. LeBoeuf, PhD, Department of Medicine, 815 Mercer St, University of Washington, Seattle, WA 98109-8050. E-mail leboeuf{at}u.washington.edu

Received October 22, 2004; revision received December 22, 2004; accepted December 29, 2004.

	Abstract

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Background— Myeloperoxidase (MPO) colocalizes with macrophages in the human artery wall, and its characteristic oxidation products have been detected in atherosclerotic lesions. Thus, oxidants produced by the enzyme might promote atherosclerosis. However, macrophages in mouse atherosclerotic tissue do not express MPO. Therefore, mice are an inappropriate model for testing the role of MPO in vascular disease. To overcome this problem, we generated and studied transgenic (Tg) mice that contained the human MPO gene.

Methods and Results— We produced human MPO-Tg mice with use of a Visna virus promoter. To confine MPO expression to macrophages, we lethally irradiated LDL receptor–deficient mice and repopulated their bone marrow with cells from wild-type mice or MPO-Tg mice. Despite having similarly high levels of cholesterol after maintenance on a high-fat, high-cholesterol diet, the MPO-Tg animals developed a 2-fold greater atherosclerotic area in the aorta than did mice transplanted with wild-type bone marrow (P=0.00003).

Conclusions— Our observations indicate that expression of human MPO in macrophages promotes atherosclerosis in hypercholesterolemic mice, raising the possibility that the enzyme might be a potential therapeutic target for preventing cardiovascular disease in humans.

Key Words: lipoproteins • myeloperoxidase • oxidative stress • atherosclerosis

	Introduction

Top Abstract Introduction Methods Results Discussion References

 
Atherosclerosis, the leading cause of premature death in Western societies, is a chronic inflammatory disease.¹ Clinical, genetic, and epidemiological evidence indicates that elevated levels of LDL are one important risk factor for the disorder. However, lipid-laden foam cells derived from macrophages are the pathological hallmark of atherosclerosis, and native LDL fails to convert cultured macrophages to such cells in vitro,² suggesting that the lipoprotein must be modified to promote vascular disease.

Oxidative pathways appear to be one important mechanism for modifying LDL, because a wide variety of structurally unrelated antioxidants inhibit atherosclerosis in animal models of hypercholesterolemia.^3,4 In vitro and in vivo studies have implicated a number of potential oxidative pathways, including those dependent on redox-active metal ions,^5–7 ceruloplasmin,⁸ 15-lipoxygenase,⁹ and nitric oxide synthase.¹⁰ The physiological relevance of these pathways to the pathogenesis of human vascular disease remains poorly understood, however.

We have proposed another potential mechanism for LDL oxidation in humans. It involves myeloperoxidase (MPO), a heme protein expressed at high levels in neutrophils, monocytes, and some populations of human macrophages.^11–13 With H₂O₂, which phagocytes also produce, MPO generates a wide array of reactive intermediates.^4,11,12 Although these species are important for host defense,¹² oxidizing intermediates also have the potential to damage inflamed tissue.¹⁴

LDL that has been modified in vitro by the MPO-peroxide system is taken up by macrophage scavenger receptors.^15–17 Importantly, MPO is found in human atherosclerotic tissue, where it colocalizes with macrophages.^11,13 Immunohistochemical studies with antibodies thought to be specific have detected the enzyme’s oxidation products in human atherosclerotic lesions.¹⁸ Moreover, mass spectrometry has detected protein and lipid oxidation products characteristic of MPO in human lesions and lesion LDL.^19–22 Collectively, these observations provide strong evidence that MPO is present and enzymatically active in human atherosclerotic tissue and that LDL is one of its targets.

Subsequently, MPO has been proposed to play a number of other roles in vascular disease. In subjects with established coronary artery disease, blood levels of MPO are elevated,²³ and they predict the risk of myocardial infarction in subjects with unstable angina.²⁴ A promoter polymorphism that lowers MPO expression in vitro is associated with a decrease in risk of clinical events in patients with coronary artery disease.²⁵ Moreover, MPO is taken up by cultured endothelial cells and has been detected in endothelial cells in vivo, suggesting that it could contribute to endothelial dysfunction.²⁶ It might also reverse the cardioprotective effects of HDL, because recent evidence suggests that it oxidizes HDL in humans.^27,28

Although mouse models have provided important insights into the pathogenesis of atherosclerosis, macrophages in murine atherosclerotic tissue do not express immunoreactive MPO on Western blots.²⁹ Moreover, only low levels of the enzyme’s characteristic products are detectable by mass spectrometry in mouse atherosclerotic tissue. Thus, although mice deficient in MPO develop greater atherosclerosis, wild-type (WT) and MPO-deficient mice appear to be inappropriate models for testing MPO’s role in atherosclerosis. We therefore developed transgenic (Tg) mice that express human (h-) MPO under the control of the Visna virus long terminal repeat (LTR) promoter, which is mainly active in macrophages. In the current studies, we used these animals to determine the effect of macrophage-specific MPO expression on the development of atherosclerosis in a hyperlipidemic mouse model.

	Methods

Top Abstract Introduction Methods Results Discussion References

 
MPO Transgene
The full-length cDNA for h-MPO was amplified with use of cDNA synthesized from the HL-60 cell line poly(A⁺)RNA as a template (Clontech). The HL-60 cell line is derived from a human promyelocytic leukemia cell that expresses MPO at high levels. The primers used to amplify the cDNA had unique restriction enzyme cut sites engineered into the 5' and 3' ends, EcoRI and BamHI, respectively. The 5' primer was 5'-CGG AAT TCG ACC ATG GGG GTT CCC TTC TTC TCT-3', which includes the transcriptional start site. The 3' reverse primer was 5'-CGG GAT CCC TAG GAG GCT TCC CTC CAG GA-3', which includes the stop codon. The high-fidelity, proofreading Pfu DNA polymerase was used to amplify the cDNA (Stratagene) during the following thermocycles: 94°C for 1 minute; 30 cycles at 94°C for 30 seconds, 58°C for 45 seconds, and 72°C for 4 minutes; and a final extension for 10 minutes at 72°C. After polymerase chain reaction (PCR), the cDNA was cut with the EcoRI and BamHI enzymes overnight at 37°C.

The Visna promoter (provided by Dr Janice Clements, Johns Hopkins University)³⁰ was excised from the pGem-T vector with ApaI and NotI and subcloned into the pcDNA3.1(–) expression vector (Invitrogen). The MPO cDNA was then subcloned into the EcoRI and BamHI sites of the pcDNA3.1(–)/Visna vector. The transgene was excised for microinjection with SacII and XmnI.

Animals
Tg mice were generated directly in C57BL/6J mice, and 2 separate lines were developed. Line 1 was used for bone marrow transplant studies. LDL receptor–deficient (LDLR^–/–) and WT C57BL/6J (MPO-WT) mice, 6 to 8 weeks of age, were purchased from Jackson Laboratory (Bar Harbor, Me). MPO-deficient (MPO^–/–) animals were provided by Dr Aldons Lusis (University of California, Los Angeles) and have been described previously.²⁹

The mice were maintained in a temperature-controlled (25°C), modified specific pathogen-free facility with a strict 12-hour light/dark cycle and given free access to food and water. Mice were fed either rodent chow (Wayne Rodent BLOX 8604, Teklad Test Diets) or a Western-type diet containing 21% butterfat and 0.15% cholesterol (diet No. 88137, Harlan Teklad). The Animal Care and Use Committee of the University of Washington approved this project.

Study Design
To assess the effect of MPO on the development of atherosclerosis, we transplanted bone marrow from male mice that differed only in their MPO genotype. The recipient mice were deficient in the LDL receptor (LDLR^–/–), a hyperlipidemic mouse model.³¹ Transplantation was accomplished by irradiating female LDLR^–/– recipients with 950 rads to ablate their endogenous bone marrow. The marrow was then repopulated with bone marrow from 1 of 3 donor groups: male C57BL/6J (WT), h-MPO-Tg, and MPO^–/– (n=14 for each group). Approximately 10⁷ donor bone marrow cells were injected into the tail vein of the LDLR^–/– recipients. The bone marrow was allowed to engraft for 4 weeks, and during that time, the mice were fed irradiated standard rodent chow and kept under specific pathogen-free conditions. Mice were then fed a Western-type diet (21% fat and 0.15% cholesterol) for 13 weeks. On the morning of sacrifice, the mice were fasted for 4 hours, bled from the retro-orbital sinus, and killed by cervical dislocation. The mice were then perfused with sterile phosphate-buffered saline (PBS), and tissues were removed for analyses. Final animal numbers in groups were reduced because of animal death and inappropriate sectioning of aortic tissue.

Analyses of Plasma
Plasma total cholesterol levels were determined with a colorimetric kit (Diagnostic Chemicals Ltd) with cholesterol standards (Sigma).³² Plasma triglyceride levels were determined calorimetrically after removal of free glycerol (Roche Diagnostics). Plasma lipoproteins were separated by high-resolution size-exclusion chromatography (Superose 6 column, Amersham Biosciences). A 100-µL aliquot of plasma pooled from 3 mice was separated at a flow rate of 0.2 mL/min with PBS. Aliquots (100 µL) from each 0.5-mL fraction were used for cholesterol determinations.

Quantification of Atherosclerosis
Atherosclerosis was evaluated by analyzing serial sections at the aortic root and by en face analysis of the aortic arch. Lesion sizes were quantified in the aortic root essentially as described.³³ In brief, the upper sections of the hearts were fixed overnight in 10% neutral-buffered formalin and embedded in paraffin the following day. Aortic root lesion area was quantified, beginning at the termination of the aortic valve and spanning 400 µm of the ascending aorta. Every other section (5 µm thick) through the aortic root was measured for analysis. A subset of 5 sections from each animal spanning the region were stained with Movat’s stain³⁴ to quantify lesion area (Image Pro Plus, Media Cybernetics). For the en face analysis, aortas were cut longitudinally, pinned flat on black wax, and photographed. Total surface area and lesion area in sections of ascending and thoracic aorta 10 mm long were quantified.

Western Blotting
Total protein was extracted from cells or tissue by homogenization in lysis buffer (1% NP-40, 0.5% sodium deoxycholate, and 0.1% sodium dodecyl sulfate in PBS) supplemented with 0.1% hexadecyltrimethyl ammonium bromide.¹¹ After separation by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, protein was electrophoretically transferred to a nitrocellulose membrane and incubated in blocking solution (Tris-buffered saline with 0.05% Tween-20 and 8% nonfat dry milk) overnight at 4°C. All antibodies were diluted in blocking solution. MPO was detected with a polyclonal rabbit antibody to h-MPO (BioDesign, 1:3000 dilution). The secondary antibody was a polyclonal donkey antibody to rabbit IgG conjugated to horseradish peroxidase (1:2000 dilution). Immunoreactive protein was detected with chemiluminescence (ECL, Amersham Biosciences).

Peroxidase Activity
Peritoneal cells were harvested with ice-cold PBS (pH 7.4) 72 hours after injection of 1 mL of 4% thioglycollate. Cells were then pelleted by centrifugation (450g, 5 minutes, 4°C), resuspended in 155 mmol/L NH₄Cl, and incubated at 37°C for 10 minutes to lyse contaminating red blood cells. Cells (2x10⁶) were then plated in 60-mm dishes and allowed to adhere for 2 hours in Dulbecco’s modified Eagle’s medium with 10% fetal bovine serum supplemented with 2 mmol/L glutamine. The medium was removed, and the cells were washed twice with PBS. Adherent macrophages were lysed in PBS containing 1% Triton X-100. Protein concentrations were determined with bicinchoninic acid. Peroxidase activity was determined from the initial rate of tetramethylbenzidine (TMB) oxidation (K-Blue MAX, Neogen Corp) under conditions where oxidation was a linear function of protein concentration. One unit of peroxidase activity was defined as a change in absorbance of 1 at 650 nm at 15 minutes and was normalized for protein content. The detection limit of this assay was <3 mU.

Immunohistochemistry
Sections were deparaffinized in xylene and rehydrated through graded ethanol to distilled water. Endogenous peroxidase activity was quenched by incubating slides in 3% H₂O₂ in methanol for 30 minutes. Slides were rinsed in 2 changes of PBS, pH 7.4, and then incubated in PBS with 0.3% Triton X-100 for 30 minutes at room temperature. Sections were blocked according to the manufacturer’s instructions (Histomouse kit, 95-9541, Zymed Laboratories, Inc). Primary antibodies were diluted in PBS with 0.05% Tween 20 at the following concentrations: rabbit polyclonal anti–h-MPO, 1:400 (Biodesign International), and rat monoclonal anti–mac-3, 1:200 (PharMingen). The slides were next incubated with the appropriate secondary antibody conjugated to biotin diluted 1:200 and then with streptavidin–horseradish peroxidase (Zymed Histomouse kit). Color was developed with 3-amino-9-ethylcarbazole (AEC) as the substrate.

Bone Marrow Engraftment
Bone marrow genomic DNA was isolated from experimental mice when the mice were humanely killed. To assess bone marrow engraftment, 10 µg of bone marrow DNA from recipient mice was compared with a standard curve (from 0% to 100%) of DNA from male C57BL/6J mice. DNA was digested with EcoRI, separated on a 1% agarose gel, and then Southern-blotted. Blots were hybridized with a radiolabeled 321-bp PCR-generated fragment of the male-specific SRY (sex-determining region of the Y chromosome) gene. The primers used to generate the probes were (forward) 5'-GCA TTT ATG GTG TGG TCC CGT G-3' and (reverse) 5'-CCA GTC TTG CCT GTA TGT GAT GG-3'. Bands were quantified with use of the Cyclone Phosphor Imager (Packard Instrument Co). To estimate bone marrow engraftment, band intensity from experimental animals was plotted on the standard curve of male DNA.

Reverse Transcription–PCR
Tissue expression of h-MPO transgene mRNA was evaluated by reverse transcription (RT)–PCR. Total RNA was prepared from mouse tissue with Tripure reagent (Roche Diagnostics) and resuspended in 20 µL of deionized water. cDNA was synthesized from 5 µg of total RNA with Moloney-Murine Leukemia Virus (M-MLV) RT (Promega). PCR amplification was performed with primers specific for h-MPO (forward, 5'-CCC GCA TCA AGA ACC AAG-3'; reverse, 5'-GGT GAG GAG ACA GGG GTC A-3'), and it yielded a 269-bp product. PCR conditions were as follows: (95°C for 1 minute, 68°C for 5 minutes) and a final elongation at 68°C for 7 minutes.

Statistical Analyses
Data are reported as the mean±SEM. Differences between group means were evaluated by the Student t or the Mann-Whitney U test. P0.05 was considered statistically significant.

	Results

Top Abstract Introduction Methods Results Discussion References

 
Generation of h-MPO-Tg mice
A subcloned Visna virus promoter containing the entire 3LTR was ligated into pcDNA 3.1. MPO cDNA that had been amplified by PCR from the human leukemia cell line HL60 was inserted into pcDNA 3.1 (Figure 1A). The Visna virus promoter–h-MPO fusion gene was isolated, purified, and sequenced. Chinese hamster ovary (CHO) cells transfected with the fusion gene, but not WT cells, expressed protein of 80 kDa that reacted with an antibody monospecific for h-MPO (Figure 1B). This is consistent with expression of a functional, partially processed monomeric form of recombinant h-MPO reported in the literature.^35–37 Detergent extracts of CHO cells transfected with the fusion gene demonstrated 3-fold increased peroxidase activity, strongly suggesting that they were synthesizing enzymatically active MPO (0.31 and 0.69 mU/µg protein for 2 WT CHO colonies and 1.62 and 1.41 mU/µg for 2 independently transfected CHO cells).

View larger version (56K): [in this window] [in a new window]   Figure 1. Characterization of h-MPO-Tg mice. A, Schematic of transgene used to make h-MPO-Tg mice. Expression of h-MPO cDNA was driven by Visna virus LTR. PolyA signal from cloning vector was also included. B, Two sets of CHO cells transfected with MPO transgene (CHO/MPO) expressed an 80-kDa protein that was immunoreactive with an antibody to h-MPO (rMPO), whereas untransfected (CHO/WT) cells did not. Each lane contained 20 µg total cellular protein. C, DNA was isolated from WT (C57BL/6J) and Tg mice of lines 1 and 2, digested PstI, and Southern-blotted with probe for the transgene. D, mRNA isolated from each line of h-MPO-Tg mice and WT controls was subjected to RT-PCR with primers for the transgene. Both lines of h-MPO-Tg mice expressed transgene in all tissues examined. No signal was detected in WT C57BL/6J mice. Negative control was no template; positive control was h-MPO transgene DNA. BGH indicates bovine growth hormone.

To generate the Tg mice, the fusion gene was injected into male pronuclei of fertilized eggs derived from C57BL/6J mice. Resulting Tg mouse lines (lines 1 and 2) carried 50 (line 1) and 10 (line 2) copies of the transgene, as determined by slot blotting against increasing amounts of C57BL/6J genomic DNA and detected by Southern blotting (Figure 1C). Mice from line 1 were used in the studies described next. The h-MPO-Tg mice reproduced normally, weighed the same as WT C57BL/6J mice, and appeared healthy. Studies with line 2 mice gave similar results (data not shown). The mice were maintained in a hemizygous state for the MPO transgene, so that insertional mutagenesis of the mouse genome would be unlikely to contribute to the observed phenotypes.

Macrophages of h-MPO-Tg Mice Express Functional MPO
The Visna virus LTR directs expression in a number of tissues, including macrophages, brain, heart, lymphocytes, lung, and muscle. RT-PCR revealed expression of h-MPO in all of the tissues analyzed from the h-MPO-Tg mouse line but not in those from the WT mice (Figure 1D). Western blotting detected h-MPO protein with an apparent molecular weight of 80 kDa in thioglycollate-elicited peritoneal macrophages from the h-MPO-Tg mice but not in macrophages from the WT or MPO-deficient mice (Figure 2A). Importantly, peroxidase activity was 2-fold higher in peritoneal macrophages isolated from the h-MPO-Tg mice than in those from the WT controls, suggesting that the Tg macrophages expressed active peroxidase (Figure 2B).

View larger version (17K): [in this window] [in a new window]   Figure 2. Macrophages from h-MPO-Tg mice express functional MPO. A, Immunoblot analysis of mouse peritoneal macrophages harvested 96 hours after thioglycolate injection, with an antibody monospecific for h-MPO. Each lane was loaded with 165 µg total protein. Cells from both lines of h-MPO-Tg mice expressed 80-kDa recombinant monomeric MPO (rMPO), whereas MPO-deficient (MPO KO) and WT (C57BL/6J) cells did not. B, Peroxidase activity of mouse peritoneal macrophages harvested 72 hours after injection of thioglycollate. Macrophages were isolated by adhesion from peritoneal cells. Peroxidase activity was assessed with TMB in lysates of the isolated macrophages. n=3 for each group. *P=0.02 and **P=0.005, vs WT animals.

LDLR^–/– Mice Transplanted With h-MPO-Tg Bone Marrow Develop Larger Atherosclerotic Lesions Than Do Those Transplanted With WT Bone Marrow
Because RT-PCR detected the h-MPO transgene in a wide range of tissues in the h-MPO-Tg mice, these animals were not suitable for examining the role of macrophages in atherosclerosis. To obtain mice that expressed the transgene selectively in macrophages, we infused bone marrow from male donor h-MPO-Tg or WT mice into lethally irradiated WT female LDLR^–/– mice. Bone marrow engraftment was equivalent in the 2 groups of recipient animals, as determined by nearly identical band intensities for SRY on Southern blots (data not shown).

Four weeks after transplantation, we placed both groups of LDLR^–/– mice (h-MPO-TgLDLR^–/– and WTLDLR^–/–) on a Western-type diet lacking cholate. After 13 weeks on the high-fat diet, the average size of the atherosclerotic lesion at the aortic sinus (Figure 3A) was 2.3-fold greater in the h-MPO-TgLDLR^–/– mice than in the mice transplanted with WT bone marrow (mean±SD, 297 300±28 000 versus 131 000±12 200 µm²/section, P=0.00003). Additionally, the mice transplanted with h-MPO-Tg bone marrow cells developed 2.8-fold larger lesions in the aortic arch than did the WT transplanted controls, as assessed by en face quantification (1.1±0.3% versus 0.4±0.1% surface area, P=0.05).

View larger version (19K): [in this window] [in a new window]   Figure 3. Atherosclerotic lesion area in aortic root and plasma lipid levels in LDLR–/– mice subjected to bone marrow transplantation. A, Bone marrow from MPO WT, h-MPO-Tg, or MPO-deficient (MPO KO) mice was transplanted into lethally irradiated LDLR–/– mice. Four weeks after transplantation, animals were placed on Western diet for 13 weeks. Lesion area in aortic sinus was assessed as described in Methods. *P=0.00003, **P=0.0006, vs WT mice. Lesion area between h-MPO-Tg and MPO-KO mice was not significantly different. B, Plasma cholesterol and triglyceride levels of bone marrow–transplanted mice fed Western diet for 13 weeks. No significant differences were found between mice transplanted with bone marrow cells from WT mice and h-MPO-Tg or MPO-deficient mice.

To address the possibility that lesion development in the h-MPO-TgLDLR^–/– mice might have been accelerated because of higher plasma lipid levels, we measured plasma total cholesterol and triglyceride levels (Figure 3B). However, levels of both mouse groups were similar, regardless of whether the LDLR^–/– mice had been transplanted with marrow derived from h-MPO-Tg or WT mice. The distribution of cholesterol and triglyceride values among lipoprotein fractions, as assessed by fast protein liquid chromatography, showed similar profiles between the transplantation groups, and total plasma cholesterol levels failed to show any correlation with lesion size (data not shown).

LDLR^–/– Mice Transplanted With h-MPO-Tg Bone Marrow Develop Advanced Atherosclerotic Lesions
Histochemical analysis of aortic root tissue harvested from both h-MPO-TgLDLR^–/– and WTLDLR^–/– mice demonstrated complex lesions, characterized by a disrupted endothelium, abundant proteoglycans and matrix proteins, fibrous caps, areas of necrosis, and evidence of cholesterol crystals (Figure 4A and 4B). Although lesion sizes differed between study groups, immunohistochemical staining with an antibody specific for macrophages revealed no major differences in the numbers or distribution of macrophages in the lesions. These observations suggest that expression of the h-MPO transgene in macrophages had little effect on lesion composition in LDLR^–/– mice fed the Western-type diet for 13 weeks.

View larger version (110K): [in this window] [in a new window]   Figure 4. Immunohistochemistry analysis of atherosclerotic lesions from LDLR–/– mice. Movat staining of aortic lesion from LDLR–/– mice transplanted with bone marrow from WT (A) and h-MPO-Tg (B) mice (magnification x4). Immunohistochemical staining for h-MPO of aortic lesions of WT (C) and h-MPO-Tg (D) mice. MPO-positive cells were detected in intima and adventitia (closed arrows) of h-MPO-Tg mice only. Diffuse MPO staining in lesions was also observed in intima (open arrow). Macrophages (Mac-3) staining of aortic lesions of WT (E) and h-MPO-Tg (F) mice. Positive immunohistochemical stain for macrophages is indicated by red immunoreaction product. Magnification x20.

We tested whether h-MPO protein and mRNA were present in the mouse lesions. Using a polyclonal antibody to h-MPO, we found no MPO-positive immunostaining cells in any aortic sections taken from LDLR^–/– mice transplanted with cells from WT mice (Figure 4C). In contrast, MPO-positive cells and diffuse extracellular staining were seen in intimal lesions of aortas from LDLR^–/– mice transplanted with cells from h-MPO-Tg mice (Figure 4D). Occasional adventitial tissue also reacted with the antibody to MPO. mRNA corresponding to the h-MPO transgene was detected in these mice by RT-PCR of RNA isolated from aortas (Figure 5). Immunostaining of adjacent sections of tissue with the mac-3 antibody indicated that MPO-positive cells colocalized with macrophages, strongly suggesting that the protein was being expressed by these phagocytes (Figure 4E and 4F).

View larger version (30K): [in this window] [in a new window]   Figure 5. RT-PCR for MPO transgene with aortic RNA. Aortic tissue was processed as described in Methods, and RT-PCR was used to test for presence of h-MPO mRNA. mRNA for h-MPO was detected in LDLR–/– mice transplanted with bone marrow from h-MPO-Tg mice but not WT (C57BL/6J) mice. Negative control was done with no template, and positive control was done with Tg plasmid.

Effect of MPO Deficiency on Atherosclerosis
Previous studies of 2 different genetic models of hypercholesterolemia have suggested that atherosclerosis is also enhanced in MPO-deficient mice.²⁹ To confirm these observations, we transplanted marrow from MPO^–/– mice into lethally irradiated LDLR^–/– mice, allowed the animals to recover for 4 weeks, and then fed them the Western-type diet. After 13 weeks on the high-fat diet, the average size of the atherosclerotic lesion at the aortic sinus (Figure 3A) was 1.9-fold greater in the MPO^–/–LDLR^–/– mice than in the mice transplanted with WT bone marrow (mean, 243 000±25 300 versus 131 000±12 200 µm² per section, P=0.0006). Mice transplanted with marrow from WT mice and the MPO^–/– mice had similar plasma cholesterol and triglyceride levels (Figure 3B). These observations confirm that atherosclerosis increases in hyperlipidemic mice when their phagocytes lack MPO, strongly suggesting that neutrophils and monocytes play a role in atherogenesis that is distinct from that of macrophage-associated MPO in the artery wall.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
Although MPO is an important microbicidal component of innate immunity, its characteristic products have been detected in human atherosclerotic tissue and lesion LDL, suggesting that it might also damage tissue during inflammation.¹⁴ Because mouse atherosclerotic lesions are devoid of immunoreactive MPO and 3-chlorotyrosine,²⁹ a specific product of MPO, we developed mice overexpressing MPO as driven by the Visna virus promoter. To obtain macrophage-specific expression, we used bone marrow cells from h-MPO-Tg or WT mice to repopulate irradiated LDLR^–/– mice. We hypothesized that mice overexpressing h-MPO in their macrophages would develop more severe atherosclerosis than control mice. In support of this hypothesis, we found that mice repopulated with h-MPO-Tg bone marrow developed significantly larger lesions than did mice transplanted with WT marrow. Thus, expression of h-MPO in macrophages promoted the development of atherosclerotic lesions in mice.

Only a subset of macrophages in human atherosclerotic lesions produces MPO,^11,13 suggesting that certain cells respond to signals that induce MPO expression.¹³ We observed immunoreactive MPO protein in a subset of macrophages in atherosclerotic lesions and in the adventitial layer of the mice transplanted with h-MPO-Tg cells. This MPO expression pattern was similar to that observed in early human lesions.^11,13 In contrast, we were unable to detect any cells that reacted with antibody to h-MPO in lesions of LDLR^–/– mice transplanted with WT marrow, as other observers have noted.²⁹ These data suggest that MPO expression in a subset of macrophages is sufficient to promote the development of atherosclerotic lesions in mice. Alternatively, expression of h-MPO may have been more robust earlier in lesion development.

The different levels of atherosclerosis in the various groups of mice could have resulted from Tg effects on plasma lipid levels. Importantly, the LDLR^–/– mice transplanted with h-MPO-Tg, WT, or MPO^–/– bone marrow had similar plasma lipid levels and lipoprotein profiles. Thus, abnormalities in lipid metabolism are not likely to explain the increase in atherosclerosis seen in the mice with macrophage-specific h-MPO-Tg expression.

We observed a significant effect on lesion development in LDLR^–/– mice transplanted with h-MPO-Tg bone marrow–derived cells, together with expression of immunoreactive protein in a subset of macrophages in the artery wall. Paradoxically, lesions are also larger in mice transplanted with bone marrow cells derived from MPO-deficient mice.²⁹ MPO-deficient mice also develop more extensive pathology than do WTs in models of ischemia/reperfusion injury³⁸ and multiple sclerosis³⁹ and are susceptible to pathogen infections.^40,41 It is important to note that neutrophils and monocytes are the major sites of MPO expression in humans and mice. Thus, expression of MPO in these cells appears to be protective against a variety of inflammatory diseases. One important mechanism may be the role of MPO in innate immunity. Indeed, chronic inflammation is thought to be of central importance in atherogenesis,¹ and both mice and humans deficient in MPO are susceptible to infection with microbial pathogens.^12,40,41

Neutrophils and monocytes isolated from mice have low levels of MPO relative to the same populations of cells isolated from humans.⁴² This finding is in line with previous observations that macrophages in WT mouse atherosclerotic tissue do not express immunoreactive MPO on Western blots and that only low, background levels of the enzyme’s characteristic products are detectable by mass spectrometry in mouse atherosclerotic tissue.²⁹ In MPO-deficient mice, the lack of protective oxidants from MPO may result in inflammatory or immunologically driven acceleration of lesion formation.⁴³ Conversely, overexpression of MPO at the artery wall could increase the level of oxidants produced by MPO and might lead to pathological damage. Collectively, these observations suggest that mice are sensitive to alterations in MPO expression in phagocytic white blood cells and that the increased level of expression seen in our Tg animals resulted in a significant increase in atherosclerosis.

In summary, we have demonstrated that expression of h-MPO in murine macrophages increases atherosclerosis in LDLR^–/– mice fed a high-fat diet. Our observations support the hypothesis that macrophage-specific expression of MPO is atherogenic and raise the possibility that lipoprotein oxidation is one important mechanism. They also suggest that MPO might be a promising target for new drugs aimed at preventing cardiovascular disease. In future studies, it will be of interest to determine whether MPO in blood and endothelial cells also contributes to atherogenesis and endothelial dysfunction.

	Acknowledgments

 
We thank Dr A.J. Lusis (University of California at Los Angeles) for providing MPO^–/– mice, Dr J. Clements (Johns Hopkins University) for providing the Visna virus promoter, Dr C. Ware (University of Washington) for aid in generation of h-MPO-Tg mice, and Shoshana Maslan and Angelique Nelson for technical assistance. The Transgenic Resource Facility (Department of Comparative Medicine, University of Washington) and the Nathan Shock Center of Excellence for the Basic Biology of Aging (National Institutes of Health [NIH]/National Institute on Aging [NIA] AG13280) helped generate the MPO-Tg mice. This project was supported by grants from the NIH (HL52848, HL64344, AG021191, NIA T32 AG00057) and the Donald W. Reynolds Foundation. Funding for Tim McMillen was provided by a Genetic Approaches to Aging Research Training Grant (NIH/NIA T 32 AG00057).

	References

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