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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2000;20:2100.)
© 2000 American Heart Association, Inc.

Atherosclerosis and Lipoproteins

Overexpression of 15-Lipoxygenase in Vascular Endothelium Accelerates Early Atherosclerosis in LDL Receptor–Deficient Mice

Dror Harats; Aviv Shaish; Jacob George; Mary Mulkins; Hiroki Kurihara; Hana Levkovitz; Elliott Sigal

From the Institute of Lipid & Atherosclerosis Research (D.H., A.S., J.G., H.L.), Sheba Medical Center, Tel-Hashomer, Israel; Bristol-Myers Squibb (M.M., E.S.), Princeton, NJ; and the Third Department of Medicine (H.K.), University of Tokyo, Hongo, Tokyo, Japan.

Correspondence to Dror Harats, MD, Institute of Lipid & Atherosclerosis Research, Sheba Medical Center, Tel-Hashomer, 52621 Israel. E-mail dharats{at}post.tau.ac.il

	Abstract

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Abstract—To study the possible role of the human lipid-oxidizing enzyme 15-lipoxygenase (15-LO) in atherosclerosis, we overexpressed it specifically in the vascular wall of C57B6/SJL mice by using the murine preproendothelin-1 promoter. The mice overexpressing 15-LO were crossbred with low density lipoprotein (LDL) receptor–deficient mice to investigate atherogenesis. High levels of 15-LO were expressed in the atherosclerotic lesion in the double-transgenic mice as assessed by immunohistochemistry. The double-transgenic, 15-LO–overexpressing, LDL receptor–deficient mice (LDLR^-/-/15LO) developed significantly larger atherosclerotic lesions at the aortic sinus compared with lesions in the LDL receptor–deficient (LDLR^-/-) mice after 3 and 6 weeks (107 000 versus 28 000 µm² [P<0.001] and 121 000 versus 87 000 µm² [P<0.05], respectively) of an atherogenic diet. LDL from the LDLR^-/-/15LO mice was more susceptible to oxidation than was the LDL from the control LDLR^-/- mice, as shown by a shorter lag period for copper-induced conjugated diene formation. On the other hand, no differences were found in the levels of serum anti–oxidized LDL antibodies between the study groups. There were also no differences with respect to the density of macrophages and T lymphocytes infiltrating the lesions in both experimental groups. Taken together, these results support the hypothesis that 15-LO overexpression in the vessel wall is associated with enhanced atherogenesis.

Key Words: 15-lipoxygenase • oxidation • endothelium • gene expression • atherosclerosis

	Introduction

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Early atherosclerotic lesions are characterized by lipid-laden foam cells in the arterial intima.¹ There is now ample evidence to suggest that LDL oxidation occurs in vivo in the vicinity of atherosclerotic plaque.^{2 3} Many mechanisms, enzymatic and nonenzymatic, have been proposed for the oxidation of LDL in the atherosclerotic plaque. Cellular lipoxygenases (LOs) have been suggested to be involved in LDL modification by endothelial cells and macrophages.^{4 5} 15-LO has been suggested to be a mediator of oxidation in vivo,⁶ although other enzymes, such as myeloperoxidase, have been suggested as well.⁷

Human and rabbit 15-LOs, as well as the leukocyte-type 12-LO, are unique in their ability to oxidize fatty acids esterified to membranes and LDL.^{4 8 9} The 15-LO enzyme forms hydroperoxy derivatives of linoleic acid (13-hydroperoxyoctadecadienoic acid) and arachidonic acid (15-hydroperoxyeicosatetraenoic acid) and is induced in atherosclerotic plaques.¹⁰

Several lines of evidence suggest the involvement of 15-LO in LDL oxidation. It has been implicated in the oxidative modification of LDL in cultured endothelial cells and in monocytes.^{4 11} Soybean 15-LO incubated with LDL in the presence of phospholipase A₂ oxidizes LDL, which is recognized by the scavenger receptor.¹² Moreover, human 15-LO has been shown to oxidize LDL without requiring a phospholipase,¹³ and fibroblasts that overexpress 15-LO generate minimally modified LDL with bioactive properties.¹⁴ The enzyme expression and activity in rabbit and human lesions^{15 16 17 18 19} provide the most convincing evidence for the localization of the enzyme in the atherosclerotic lesion. Moreover, elevated activity was found throughout the aortas of Watanabe and cholesterol-fed rabbits, suggesting that the enzyme may be a response to hypercholesterolemia.²⁰ Hence, 15-LO may be induced in the vessel wall early in atherogenesis before plaque is formed.

Because 15-LO is expressed in the vascular wall, it can promote atherogenesis by altering endothelial cell function. Several works indicate that 15-LO is proatherogenic. It has been implicated in endothelial cell oxidation of LDL,⁴ and the enzyme metabolites have been shown to cause injury to cultured endothelial cells,²¹ to induce the expression of adhesion molecules on human umbilical vein endothelial cells,²² and to bring about the appearance of LDL oxidation products in rabbit iliac arteries.²³ Moreover, a recent study shows that disruption of the 12/15-LO gene diminishes atherosclerosis in apoE-deficient mice.²⁴ Sparrow and Olszewski,²⁵ who used the dual cyclooxygenase-LO inhibitor, found discrepancies between the drug concentrations required to inhibit the oxidation of free fatty acid and those required to inhibit LDL modification by murine macrophages. However, studies with 15-LO–specific inhibitors lacking significant antioxidant activity have suggested that the enzyme is involved in atherogenesis.^{26 27} In contrast to the proatherogenic effects of 15-LO, several studies report that the enzyme metabolite 13-hydroxyoctadecadienoic acid has antiatherogenic activities.^{28 29 30} The antiatherogenic activity of 15-LO was demonstrated in vivo by Shen and colleagues.^{31 32} They developed transgenic New Zealand White rabbits and heterozygous Watanabe heritable hyperlipidemic rabbits with integrated human 15-LO, driven by the lysozyme promoter. Surprisingly, although the 2 lines of transgenic rabbit showed 15-LO overexpression in macrophages, atherosclerotic lesion development was reduced in the transgenic animals.

To investigate further the relation of 15-LO to atherosclerosis in the mouse, we created vascular-specific human 15-LO–overexpressing transgenic mice by using the murine prepoendothelin-1 promoter.³³ In the present study, we used murine prepoendothelin-1 promoter for overexpressing 15-LO specifically in the vascular wall of LDL receptor–deficient (LDLR^-/-) mice and studied its effect on atherogenesis.

	Methods

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LDLR^-/-/15LO Double-Transgenic Mice
To establish a colony of LDLR^-/- mice with human 15-LO activity derived with the preproendothelin promoter (LDLR^-/-/15LO mice), we crossbred LDLR^-/- mice³⁴ with 2 independent homozygous colonies of preproendothelin-1 promoter–15-LO–overexpressing mice with a background of C57B6/SJL.³³ Double-transgenic mouse colonies were established and screened by polymerase chain reaction with use of primers for both transgenes and the wild-type LDL receptor gene as described.⁶

Experimental Design
Two sets of experiments were performed. In each experiment, forty-five 3-month-old mice in each group were studied. The mice were fed a high-cholesterol high-fat diet containing 15.75% fat (43% saturated fat), 1.25% cholesterol, and 0.5% sodium cholate (Harlan, Teklad). To minimize oxidation of cholesterol and lipids, the diet was kept in a cold room at 4°C and fed daily ad libitum. Animals from each group were euthanized at 0, 3, and 6 weeks in the first experiment and at 0, 3, and 9 weeks in the second experiment. Fifteen mice from each group were killed at each time point. The Sheba Medical Center Animal Studies Committee (Tel-Hashomer, Israel) approved all procedures.

Plasma Lipid Levels
Cholesterol and triglyceride levels were measured with an enzymatic kit (Boehringer-Mannheim) at 0, 3, 6, and 9 weeks.

Lipoprotein Oxidation
LDLs (fraction 1.019 to 1.063) were isolated from pooled plasma of 9 mice from each group, and 3 pools from each group were analyzed separately. Lipoproteins were incubated at a concentration of 50 µg/mL PBS, pH 7.4, with 15 µmol/L CuSO₄. Incubation was carried out at 37°C in the dark. Lipid oxidation was measured as conjugated diene formation at 234-nm wavelength.³⁵

Macrophage Preparation
Peritoneal macrophages were isolated and purified as previously described.³⁶ Briefly, macrophages in LDLR^-/- and LDLR^-/-/15LO mice were elicited by intraperitoneal injection of 2.4% Brewer thioglycollate medium (2 mL, Difco Laboratories). Three days later, the peritoneal macrophages were harvested by 2 mL PBS lavage. Washed cells, 1x10⁶ in a total volume of 2 mL, were plated on 35-mm-diameter polystyrene tissue-culture Petri dishes with DMEM (Biological Industries) supplemented with 20% heat-inactivated FCS. The cells were incubated for 2 hours at 37°C in 5% CO₂/95% air. Adherent macrophages were washed twice with DMEM, collected after trypsin treatment, pelleted at 2000 rpm, centrifuged for 5 minutes, and resuspended in 1 mL of cold PBS containing 5 mmol/L glucose, pH 7.4.

15-LO Enzymatic Activity
To assess the level of expression of the human 15-LO in the double-transgenic mice, we performed an enzyme activity assay in the mouse tissues and in isolated peritoneal macrophages. We measured the enzyme product, 15-hydroxyeicosatetraenoic acid (15-HETE), by a standard high-performance liquid chromatography (HPLC) technique.³³ Briefly, mice were euthanized, and their organs were harvested, trimmed of fat and connective tissue, weighed, minced, and resuspended in 1 mL of cold PBS containing 0.5 mmol/L glucose, pH 7.4. For each activity assay, 200 mg of tissue was used. The reactions were carried out in a total volume of 1 mL at 37°C for 15 minutes, with 20 µmol/L arachidonic acid used as a substrate. The reaction was terminated with 100 µL glacial acetic acid, and the lipids were extracted with 1 vol isopropyl alcohol and 1 vol chloroform. An aliquot of prostaglandin B₂ was used as an internal standard. All extracts were dried under N₂ and stored at -70°C. Extracts were reconstituted in chromatography solvent and were analyzed by reverse-phase HPLC on a chromatography system (Kontron Instruments Inc) with use of an Adsorbosil C18 column (Vydac 201TP-54; 250x5 mm, 5-µm particle size). The column was developed at a flow rate of 1.0 mL/min by an isocratic solvent system, methanol/H₂O/glacial acetic acid (850:150:0.1 [vol/vol/vol]). The eluate was monitored with a Kontron 430 HPLC detector.

Assessment of Atherosclerosis in the Aortic Sinus
Quantification of atherosclerotic fatty-streak lesions was performed by measuring the lesion size in the aortic sinus. The heart and upper section of the aorta were removed from the animals, and the peripheral fat was cleaned carefully. The upper section was embedded in OCT compound (Miles Inc) and frozen. Every other section (10 µm thick) throughout the aortic sinus (400 µm) was taken for analysis. The distal portion of the aortic sinus was recognized by the 3 valve cusps that constitute the junctions of the aorta to the heart. Sections were evaluated for fatty-streak lesions after they were stained with oil red O. Lesion areas per sections were counted with use of a grid by an observer unfamiliar with the tested specimen.

Sudan IV Staining of Aortic Lesions
The aortas were dissected from the aortic arch to the iliac bifurcation and washed 1 for hour in PBS, pH 7.4, and 0.5 mmol/L EDTA on a rotating table. The aorta was then fixed with formal-sucrose (4% paraformaldehyde, 5% sucrose, 20 mmol/L butylated hydroxytoluene, and 2 mmol/L EDTA, pH 7.4) overnight. The adventitial fat was trimmed from the aorta under a microscope and opened longitudinally, rinsed briefly in 70% ethanol, immersed for 6 minutes in a filtered solution of Sudan IV (Sigma Chemical Co) in 35% ethanol and 50% acetone for 10 minutes, and destained in 80% ethanol.³⁷ The Sudan IV–stained aortas were placed on a microscope slide and photographed. Lesion area was detected by morphometry.

Detection of Anti-OxLDL Antibodies by ELISA
Polystyrene plates with 96 wells (Nunc Maxisorp) were coated with either copper-induced oxidized LDL (oxLDL, at a concentration of 10 µg/mL in PBS) or native LDL overnight at 4°C. The plates were washed 4 times with PBS containing 0.05% Tween and 0.001% aprotinin (Sigma) and then blocked with 2% BSA for 2 hours at room temperature. Diluted (1:50) serum fractions were added in PBS containing 0.05% Tween and 0.2% BSA. The plates were incubated at 4°C overnight, the sera were washed, and alkaline phosphatase–conjugated goat anti-mouse IgG (Jackson Immuno-Research Laboratory Inc) was added (diluted 1:10 000 in PBS, 0.05% Tween, and 0.2% BSA) for 1 hour at room temperature. The plates were washed again, and 1 mg/mL p-nitrophenylphosphate (Sigma) in 50 mmol/L carbonate buffer containing 1 mmol/L MgCl₂, pH 9.8, was added as a substrate. The reaction was stopped at 30 minutes by adding 1 mol/L of NaOH. Absorbance was detected at a 405-nm wavelength in a Titertek ELISA reader (S.L.T Laboratory Instruments), and results are expressed as absorbance at 405 nm. Anti-oxLDL levels were calculated as binding to native LDL subtracted from oxLDL binding.

Immunohistochemistry
Immunohistochemical staining was performed by use of anti-CD4 (rat anti-mouse, clone H129.19 [L3T4]) and CD8a (clone S3-6.7 [Ly-2]) from PharMingen and macrophages (rat anti-mouse MCA 497 [F4/80]) from Serotec. Anti–malondialdehyde-LDL antibodies were obtained by immunization of mice with homologous malondialdehyde-LDL and performed by use of the Histomouse-SP-Bulk-Kit (Zymed-Laboratory Inc) for detection of mouse primaries on mouse tissues. The immunohistochemical studies were performed on 5-µm-thick frozen sections of the aortic sinus. The sections were fixed for 4 minutes in methanol at -20°C, followed by 10 minutes of incubation with ethanol at -20°C. The sections were then blocked with nonimmune goat serum for 15 minutes at room temperature, followed by incubation with CAS blocking reagent (Zymed) for 30 minutes at room temperature subsequent to incubation with biotinylated antibodies. After they were washed, the slides were incubated in 0.3% H₂O₂, followed by additional rinses, and developed with peroxidase streptavidin complex. Sections were counterstained with hematoxylin. Spleen sections were used as a positive control. Staining in the absence of first or second antibody was used as a negative control.

To determine the tissue distribution and cellular localization of 15-LO expression, primary organs and the aortic sinus were prepared for paraffin-embedded sections. Immunoperoxidase staining was assessed on 5-µm sections prepared from formalin-fixed paraffin-embedded tissues. Sections were deparaffinized and permeabilized with PBS containing 0.2% Nonidet P-40 detergent (Sigma). Sections were then immersed sequentially in PBS containing 0.5% BSA and 10% normal goat sera (blocking solution) for 10 minutes, followed by blocking solution containing 10 µg/mL avidin for 10 minutes, blocking solution containing 10 µg/mL biotin for 10 minutes, and primary polyclonal rabbit anti-human 15-LO antibody, raised against the native enzyme, at 1:2000 blocking solution (overnight at 4°C). The sections were then incubated with biotinylated goat anti-rabbit IgG (1:250, Vector Labs), and endogenous peroxidase activity was then quenched with 3% H₂O₂ for 5 minutes. Bound primary antibody was detected by ABC (Vector Labs), followed by the substrate, aminoethyl-carbazole (Vector Labs) or diaminobenzidine (Vector Labs), and counterstained with hematoxylin.

Statistical Analyses
All values are reported as mean±SE. Statistical analyses were performed by Student t test.

	Results

Top Abstract Introduction Methods Results Discussion References

 
15-LO Expression in the Double-Transgenic Mice
To determine the effect of 15-LO activity in the vascular wall on atherogenesis, we have produced transgenic LDLR^-/- mice overexpressing human 15-LO activity under the control of the endothelial cell–specific promoter, preproendothelin (LDLR^-/-/15LO mice). The double-transgenic mice were morphologically normal and healthy. 15-LO expression and activity in the LDLR^-/-/15LO mice were comparable to those obtained in transgenic 5.9mPPET-15-LO mice.³³ Although its activity and expression in vascular tissues were very high, nonvascular tissues showed minimal or no activity of 15-LO (Table 1). High activity of 15-LO, measured as 15-HETE production from arachidonic acid ex vivo, was found in heart extracts of LDLR^-/-/15LO mice fed chow and high-fat high-cholesterol diets, with a ratio of 4 to 10 of 15-HETE to 12-HETE (Figure 1). LO activity in heart extracts of LDLR^-/- mice was 8 times lower, with a 15-HETE to 12-HETE ratio of 0.12, indicating an activity of its wild-type 12-LO–like 15-LO activity. In contrast to the heart extract, no activity of 15-LO was detected in isolated peritoneal macrophages of both study groups (Figure 1). The only LO activity in the macrophages was that of the endogenous 12-LO–like 15-LO, as indicated by the 15-HETE to 12-HETE ratio of 0.3. Immunohistochemistry with anti-human 15-LO antibodies revealed abundant human 15-LO expression in the atherosclerotic lesion in the double-transgenic mice (Figure 2).

View this table: [in this window] [in a new window]   Table 1. 15-LO Expression in Tissue Extracts as Detected by Measuring Production of 15-LO Metabolite, 15-HETE, Ex Vivo

View larger version (20K): [in this window] [in a new window]   Figure 1. 15-LO activity in heart and isolated macrophages. 15-LO activity in LDLR-/-/15LO mice was detected by exposure of arachidonic acid to heart extract (solid line) or isolated macrophages (dashed line). The first peak is the internal standard. Cell isolation and HPLC conditions are described in Methods.

View larger version (122K): [in this window] [in a new window]   Figure 2. 15-LO expression in the atherosclerotic lesions. Immunoperoxidase staining for human 15-LO was assessed on 5-µm sections prepared from formalin-fixed paraffin-embedded aortic sinuses of LDLR-/-/15LO (A) and LDLR-/- (B) mice.

Plasma Lipid Levels
Plasma cholesterol and triglyceride levels were similar in LDLR^-/- and LDLR^-/-/15LO mice throughout the experiment. Although lower levels were detected in the double-transgenic group, a sharp increase in cholesterol levels was seen in both groups at 3 weeks of high-cholesterol high-fat diet (Table 2).

View this table: [in this window] [in a new window]   Table 2. Plasma Cholesterol and Triglyceride Levels

Susceptibility of Lipoproteins to Oxidation Ex Vivo
Because 15-LO has been suggested to oxidize lipoproteins in vivo, we measured the susceptibility of lipoproteins isolated from LDLR^-/- and LDLR^-/-/15LO mice to copper-induced ex vivo oxidation. Lipoprotein fraction (1.019 to 1.063) was isolated by ultracentrifugation, and susceptibility to copper-induced oxidation was measured by conjugated diene formation at 234 nm. The susceptibility to oxidation of lipoproteins isolated from both groups of chow-fed mice was similar (data not shown). However, after 3 weeks of a high-fat high-cholesterol diet, lipoproteins isolated from 15-LO mice were significantly (P<0.05) more susceptible to oxidation, as measured by the shorter lag phase in the conjugated diene formation kinetics, 87 minutes compared with 112 minutes in LDLR^-/- mice (Figure 3).

View larger version (19K): [in this window] [in a new window]   Figure 3. Susceptibility of lipoproteins isolated from LDLR-/- and LDLR-/-/15LO mice to oxidation in vitro. LDL (fraction 1.006 to 1.063) from fasting mice was isolated at the end of the experiment from plasma by ultracentrifugation. Oxidation reactions were carried out by exposing the lipoproteins to 15 µmol/L of CuSO4 at 37°C in PBS, pH 7.4. Lipid oxidation was monitored by the increase in absorbance at 234 nm. indicates LDLR-/- mice; , LDLR-/-/15LO mice.

Anti-OxLDL Antibody Levels
Levels of anti-oxLDL antibodies were slightly increased after short-term feeding of the high-fat diet in all experimental groups. However, no differences were evident between the LDLR^-/- and LDLR^-/-/15LO mice with respect to the levels of anti-oxLDL antibodies throughout the study (mean optical density±SD, 0.23±0.3 in the former group compared with 0.21±0.4 in the latter).

Atherosclerosis in LDLR^-/- and LDLR^-/-/15LO Mice
The overexpression of 15-LO in C57B6/SJL mice did not induce significantly more atherosclerosis than found in the wild-type C57B6/SJL; therefore, we performed 2 experiments to assay the effect of 15-LO overexpression on atherogenesis in the double-transgenic mice. Forty-five 3-month-old mice in each group were studied. The first experiment lasted for 6 weeks, and the second lasted for 9 weeks.

Atherosclerotic lesion area was measured at the aortic sinus (Figure 4). After 3 weeks of a high-fat high-cholesterol diet, the atherosclerotic lesion area in the aortic sinus was significantly (P<0.001) larger in LDLR^-/-/15LO mice than in LDLR^-/- mice (107 000 versus 28 000 µm², respectively). After 6 weeks of a high-fat high-cholesterol diet, the difference in the atherosclerotic lesion area in the aortic sinus was slightly smaller but significant (121 000 versus 87 000 µm², respectively; P<0.05). However, after 9 weeks of the atherogenic diet, the total amount of atherosclerosis at the sinus was elevated, and no significant difference was detected between the 2 groups of mice. At that time, a large area of atherosclerotic lesions was detected in the aortic arch and abdominal aorta in both mouse groups (data not shown). It is noteworthy that at 9 weeks no differences were evident between the experimental groups (196 000 versus 197 000 µm², respectively; P<0.05) with respect to the density of macrophages (macrophage content of 32±10% in the double-transgenic mice compared with 27±8% in the LDL-RD mice) or T lymphocytes (5±3 cells per lesion at the aortic sinus in the double-transgenic mice compared with 7±4 cells per lesion at the aortic sinus in the LDL-RD mice) in the sinus atherosclerotic lesions.

View larger version (19K): [in this window] [in a new window]   Figure 4. Extent of atherosclerotic lesions in LDLR-/- and LDLR-/-/15LO mice. The extent of fatty-streak lesions after staining with oil red O was measured in the aortic sinus by using a grid. Solid bars indicate LDLR-/- mice; open bars, LDLR-/-/15LO mice (P<0.05 by Student t test).

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
The human lipid-oxidizing enzyme 15-LO has been implicated in LDL oxidation, it has been shown to be induced in atherosclerotic lesions, and its oxidation product 15-HETE is the major hydroxyeicosatetraenoic acid in the lesion.^{15 16 17 18 19} The present study provides evidence for its oxidative role and demonstrates that 15-LO overexpression in the vascular wall of LDLR^-/- mice accelerates LDL cholesterol–mediated atherogenesis.

The role of LOs in atherogenesis has been studied in the apoE-deficient 12/15-LO knockout mice and LDL receptor knockout mice in the present study. The 2 models develop fatty streaks as a result of the delayed clearance of lipoproteins. Cholesterol levels in apoE-deficient mice on chow diet reach 400 to 600 mg/dL as a result of chylomicrons and VLDL remnant accumulation. These mice develop fatty streaks and fibrous plaque lesions at branch points and major vessels. LDLR^-/- mice develop mainly fatty streaks when fed a high-fat high-cholesterol diet only.³⁸ The results of the present study are in accord with the recent results obtained in apoE-deficient mice, showing that the disruption of the 12/15–LO gene diminishes atherosclerosis.²⁴ In contrast, studies by Shen and colleagues^{31 32} have shown that overexpression of 15-LO in monocytes/macrophages in New Zealand White rabbits and heterozygous Watanabe rabbits protects against atherosclerosis. In these studies, the protective effect was attributed to the platelet chemorepellant product, 13-hydroxyoctadecadienoic acid. The major differences between the studies of Shen and colleagues and the present study are the species and the localization of 15-LO expression. In the present study, 15-LO driven by the preproendothelin promoter was highly expressed in mouse endothelial cells, and no 15-LO activity was found in isolated peritoneal macrophages or other tissues. Moreover, 15-LO distribution in the lesion, as detected by immunohistochemistry (Figure 2), suggests that enzyme is highly expressed in the lesion. In the rabbit model, 15-LO driven by a lysozyme promoter is expressed specifically in monocytes/macrophages, but its expression in atherosclerotic macrophages has not been demonstrated.

Evidence indicates that 15-LO is expressed and active in endothelial cells. Weak hybridization was detected in vivo in Watanabe rabbit endothelium,¹⁵ endothelial cells can generate 15-HETE,³⁹ and inhibitors of 15-LO inhibited LDL oxidation by endothelial cells.⁴⁰ Hence, 15-LO activity in endothelial cells may play a role early in atherogenesis.

The increased susceptibility to ex vivo oxidation of lipoproteins isolated from LDLR^-/-/15LO mice may indicate that 15-LO overexpression in endothelial cells of the vessel wall exposes lipoproteins in the subendothelial space to increased oxidative stress, as it does in vitro.^{6 11 12 13} Because 15-LO is cytosolic, it has been suggested that it initiates extracellular LDL oxidation by oxidation of cell membrane lipids and that the radical membrane products could be transferred to LDL in the vicinity of the endothelial cell. The finding that LDL isolated from LDLR^-/-/15LO mice fed an atherogenic diet is more susceptible to oxidation compared with LDL isolated from LDLR^-/- mice may indicate that 15-LO overexpression in endothelial cells contributes to LDL oxidation in this model. However, the lack of difference in anti-oxLDL antibodies may suggest that there is no more oxidized LDL in the plasma of 15-LO–overexpressing mice. We speculate that the induction of 15-LO in LDL^-/-/15LO mice might accelerate atherogenesis in this mouse model by another mechanism, such as oxidative damage to endothelial cells. The intracellular enzyme could oxidize fatty acid inside the cell, which could lead to oxidation of phospholipids in the cell membrane. Alternatively, the enzyme could oxidize phospholipids in the cell membrane directly. 15-LO oxidation products have been suggested to affect many steps involved in atherosclerosis: triggering the expression of adhesion molecules,^{22 41 42} triggering chemotactic proteins,^{43 44} and affecting smooth muscle cell migration⁴⁵ and the activity of peroxisome proliferator-activated receptor-.⁴⁶ This increased process of atherogenesis can eventually result in enhanced foam cell formation and accelerated atherogenesis.

In summary, in the present study, we show that overexpression of 15-LO is associated with enhanced atherogenesis in LDLR^-/- mice and that LDL in these mice is more susceptible to ex vivo oxidation than is LDL isolated from LDLR^-/- mice.

	Acknowledgments

 
This work was supported by Binational Science Foundation grant No. 93-00193/3

Received October 13, 1999; accepted February 22, 2000.

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