This Article Abstract Full Text (PDF) Alert me when this article is cited 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 George, J. Articles by Harats, D. Search for Related Content PubMed PubMed Citation Articles by George, J. Articles by Harats, D. Related Collections Pathophysiology

(Circulation. 2001;104:1646.)
© 2001 American Heart Association, Inc.

Basic Science Reports

12/15-Lipoxygenase Gene Disruption Attenuates Atherogenesis in LDL Receptor–Deficient Mice

Jacob George, MD; Arnon Afek, MD; Aviv Shaish, PhD; Hana Levkovitz, BA; Nira Bloom, BA; Tillmann Cyrus, MD; Lei Zhao, MD, PhD; Colin D. Funk, PhD; Eliott Sigal, MD; Dror Harats, MD

Institute of Lipid and Atherosclerosis Research (J.G., A.S., H.L., N.B., D.H.) and the Institute of Pathology (A.A.), Sheba Medical Center, Tel-Hashomer, Sackler Faculty of Medicine, Tel-Aviv University, Israel; the Center for Experimental Therapeutics, University of Pennsylvania, Philadelphia (T.C., L.Z., C.D.F.); and Bristol-Myers Squibb Co, Princeton, NJ (E.S.).

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

	Abstract

Top Abstract Introduction Methods Results Discussion References

 
Background— Human 15-lipoxygenase (LO) and its murine analogue 12/15-LO are capable of directly oxidizing esterified fatty acids in lipoproteins and phospholipids. Because these oxidized products possess atherogenic properties, it was suggested that LOs may be involved in enhancing atherogenesis. Previous in vivo tests of the role of LOs in atherogenesis animal models, however, have yielded conflicting results.

Methods and Results— Aiming to study the role of the 12/15-LO in murine atherogenesis, we crossed LDL-receptor–deficient mice (LDL-R^-/-) with 12/15-LO–knockout mice and evaluated plaque formation 3 to 18 weeks after initiation of a high-fat diet. Atherosclerotic lesions were considerably reduced in the LDL-R/12/15-LO–double-knockout mice compared with LDL-R^-/- mice at 3, 9, 12, and 18 weeks, at the aortic root as well as throughout the aorta. The cellular composition of plaques from mice deficient in 12/15-LO did not differ with respect to macrophage and T-lymphocyte content compared with plaques from 12/15-LO littermates.

Conclusions— 12/15-LO plays a dominant role in promoting atherogenesis in LDL-R^-/- mice.

Key Words: lipoxygenase • atherosclerosis • lipoproteins • oxidation • cells

	Introduction

Top Abstract Introduction Methods Results Discussion References

 
Oxidative processes have been shown to play a dominant role in the initiation and progression of atherosclerosis in animal models.^1,2 Several lines of evidence support this contention. Oxidation-specific epitopes have been detected within the atherosclerotic plaque. In addition, in vitro studies have demonstrated that oxidized LDL (oxLDL) rather than native LDL is capable of promoting smooth muscle cell migration and endothelial, lymphocyte, and macrophage activation, properties that may explain its proatherogenic properties (reviewed by Heinecke³). Probably the most significant support for the oxidation hypothesis derives from studies showing that antioxidant administration reduces atherogenesis in various animal models.² Two recent studies suggest that administration of vitamin E may be capable of reducing coronary heart disease events in humans,^4,5 although in 3 other studies, it was found to be ineffective.^6–8 Indirect results have incriminated several candidates in promoting oxidative modification, such as NADPH oxidase, myeloperoxidase, some cytochrome P450 members, the mitochondrial electron transport system, xanthine oxidase, and lipoxygenases (LOs) (reviewed in Reference 3). In contrast to the relatively strong evidence for the role of oxLDL in atherosclerosis, however, there is little or no evidence regarding the systems that oxidize LDL in vivo.

LOs are a family of non–heme iron–containing dioxygenases that insert molecular oxygen into polyenoic fatty acids.⁹ This family includes the human and rabbit 15-LOs, as well as the porcine, rat, and murine leukocyte–type 12-LOs, all of which can directly oxidize esterified fatty acids in lipoproteins and phospholipids.¹⁰ The latter enzymes are also called 12/15-LO, because they are capable of producing both 12-hydroperoxy-eicosatetraenoic acid (12-HPETE) and 15-HPETE from arachidonic acid. There is a close structural resemblance between 15-LO and 12/15-LO, because minor alterations in amino acid sequence can account for the species differences in oxygen insertional specificity.¹¹

The involvement of LO in atherosclerosis is supported by indirect, as well as direct, in vivo observations. 15-LO was found to be present within atherosclerotic plaques of humans and rabbits, colocalizing with oxidation epitopes.¹² The powerful data supporting involvement of LO in atherogenesis, however, come from animal studies. Initial observations have shown that overexpression of 15-LO in rabbit arteries was associated with increased occurrence of oxLDL epitopes.¹³ We recently showed that overexpression of human 15-LO in endothelial cells of the vessel wall of LDL receptor–deficient (LDL-R^-/-) mice via preproendothelin promoter resulted in acceleration of early atherosclerotic lesions.¹⁴ Two recent studies have demonstrated that pharmacological inhibition of 15-LO resulted in attenuation of atherosclerosis in hypercholesterolemic rabbits.^15,16 Probably the most conclusive evidence for the role of 12/15-LO in atherosclerosis to date comes from the study by Cyrus et al¹⁷ showing that disruption of the 12/15-LO gene in apoE-knockout mice significantly retarded atherosclerosis initiation and progression. Data from Shen et al,¹⁸ however, indicate that overexpression of 15-LO in rabbit macrophages is associated with reduced atherogenesis, thus questioning the proatherogenic role of 15-LO.¹⁹

In the present study, we obtain further evidence for the role of 12/15-LO in murine atherogenesis by studying 12/15-LO gene disruption in LDL-R^-/- mice using 2 different types of high-fat diet in 2 laboratories (in Israel and the United States). The different experimental protocols of diet feeding are intended to induce either fatty streaks or more advanced lesions.

	Methods

Top Abstract Introduction Methods Results Discussion References

 
Generation of LDL-R^-/-/12/15-LO^-/-–Double-Knockout Mice
LDL-R–deficient mice were obtained from the Jackson Laboratory (Bar Harbor, Me). The generation of 12/15-LO^-/- and LDL-R^-/- mice was previously described.^20,21 12/15-LO^-/- mice on a hybrid C57BL/6x129/Sv (Israeli colony) or backcrossed to C57BL/6 (7 times) (University of Pennsylvania colony) background were crossbred with LDL-R^-/- mice. Mice were genotyped either by polymerase chain reaction analysis (LDL-R^-/-) or Southern blot analysis (12/15-LO) as described.²¹ All mice were kept on a 12-hour light/12-hour dark cycle, and food and water were available ad libitum. At the age of 11 to 12 weeks, LDL-R/12/15-LO–double-knockout and LDL-R^-/- mice were fed a Western-type diet. Mice were euthanized 3 to 18 weeks after initiation of the diet, and their hearts and aortas were removed for evaluation of the extent of atherosclerosis. Plasma was obtained for assessment of the lipid profile in all mice.

Diets
1. The Paigen-type diet contained 1.25% cholesterol, 7.5% casein, and 0.5% (wt/wt) sodium cholate (Harlan, Teklad Premier Laboratory Diets).

2. The Western-type diet contained 42% of calories from fat, 43% from carbohydrates, and 15% from protein (TD 96125, Harlan Teklad).

Lipid Profile
Total plasma cholesterol and triglyceride levels were determined with an automated enzymatic technique (Boehringer Mannheim). HDL cholesterol levels were determined by HDL cholesterol reagent (Sigma Chemical Co).

Lipoprotein Oxidation
Blood for lipoprotein isolation was collected in EDTA (1 mg/mL) from 3 pools from 3 mice in each group after 12 hours of fasting. LDL (density 1.019 to 1.063 g/L) was isolated from the plasma after density adjustment with KBr^-, by preparative ultracentrifugation at 50 000 rpm/min for 22 hours with a type 50 rotor. LDL preparations were washed by ultracentrifugation, dialyzed against 0.15 mol/L EDTA (pH 7.4), passed through an Acrodisc filter (0.22-µm pore size) to remove aggregates, and stored under nitrogen in the dark. Copper oxidation of LDL was performed by incubation of postdialysis LDL (1 mg protein/mL in EDTA-free PBS) with copper sulfate (10 µmol/L) for 24 hours at 37°C. Lipoprotein oxidation was confirmed by analysis of thiobarbituric acid–reactive substances (TBARS).²²

Assessment of Atherosclerosis in the Aortic Sinus
Atherosclerotic fatty-streak lesions were quantified by measurement of the lesion size in the aortic sinus as previously described.²³ 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 O.C.T. compound (Miles Inc) and frozen. Every other section (10 µm thick) throughout the aortic sinus (400 µm) was taken for analysis. Sections were evaluated for fatty-streak lesions after staining with oil red O. Lesion areas per section were counted by 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 for 1 hour in PBS (pH 7.4) and 0.5 mmol/L EDTA on a rotating table.²⁴ The aorta was then fixed with a 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.²⁵ The Sudan IV–stained aortas were assessed for lesion area by morphometry as previously described.²⁴

Immunohistochemical Analysis of Aortic Sinus Sections
Immunohistochemical stainings were performed as described²⁶ with the following primary antibodies: rat anti-mouse CD3 clone: 17A2 (PharMingen), rat anti-mouse VCAM-1 (clone 429 MVCAM.A, PharMingen), and rat anti-mouse Mac-1 (clone M1/70, Boehringer Mannheim). Slides were dried under airstream for 45 minutes, fixed in acetone for 5 minutes in -20°C, and rinsed 3 times for 5 minutes in Tris-buffered saline (TBS) containing 0.1% BSA and 0.05% Tween-20 (TBS-BSA-Tween). To reduce background signals, a 15-minute incubation with 10% nonimmune goat serum was followed by a 30-minute incubation with CAS (Zymed Laboratory) at room temperature. After blocking, the various primary antibodies diluted 1:50 in TBS-BSA-Tween, biotinylated second antibody, and streptavidin peroxidase conjugate (a kit from Zymed Laboratory) were added. The results of the Mac-1 and CD3 cells are expressed as positive nuclei/total plaque nuclei.

Statistical Analysis
Differences between groups with respect to lipid levels and plaque size and composition were made with a 2-sided Student’s t test. A value of P<0.05 was considered statistically significant.

	Results

Top Abstract Introduction Methods Results Discussion References

 
LDL-R–deficient mice maintain relatively moderate hypercholesterolemia and do not develop significant atherosclerosis on feeding a normal chow diet. Feeding a Paigen diet facilitates atherosclerosis concomitant with the elevation of cholesterol levels. The LDL-R–deficient mouse model, similar to the apoE-knockout model, displays features that indicate the involvement of oxidation in enhanced atherogenesis.

LDL-R^-/-/12/15-LO^-/- mice, like LDL-R^-/- littermates, were healthy and fertile, gaining weight in a similar manner regardless of the diet provided.

After 3 weeks and 9 weeks on the high-fat diet, LDL-R^-/-/12/15-LO^-/-–double-knockout mice and LDL-R^-/- controls (12/15-LO^+/+) maintained similar levels of cholesterol and triglycerides (Table).

View this table: [in this window] [in a new window]   Table 1. Plasma Cholesterol and Triglyceride Levels in Paigen Diet–Fed Mice

Mean cholesterol and triglyceride levels in mice fed the Western diet for 12 weeks (3 pools of 3 mice each) were not different between LDL-R^-/-/12/15-LO^-/- (1445±171 and 308±59 mg/dL, respectively) and LDL-R^-/-/12/15-LO^+/+ (1503±195 and 341±25 mg/dL, respectively) mice. Mean cholesterol and triglyceride levels in mice fed the Western diet for 18 weeks (3 pools of 3 mice each) did not differ significantly between LDL-R^-/-/12/15-LO^-/- (946±90 and 313±39 mg/dL, respectively) and LDL-R^-/-/12/15-LO^+/+ (933±91 and 280±26 mg/dL, respectively) mice.

Pooled plasma LDL from LDL-R^-/-/12/15-LO^-/- mice displayed no significant difference in susceptibility to in vitro oxidation compared with plasma from LDL-R^-/-/12/15-LO^+/+ mice (mean of 91.6 minutes in the former compared with 84.2 minutes in the latter; P=0.93).

LDL-R^-/-/12/15-LO^-/-–double-knockout mice fed a high-fat diet for 3 weeks exhibited significantly reduced early atherosclerotic lesions (9100±1700 µm²) compared with LDL-R^-/-/12/15-LO^+/+ mice (25 200±3000 µm²; P<0.05) at the aortic root. After 9 weeks of high-fat diet supplementation, atherosclerotic lesions were also reduced in LDL-R^-/-/12/15-LO^-/- mice (51 000±9000 µm²) compared with control mice (225 000±31 000 µm²; P<0.001; Figure 1).

View larger version (12K): [in this window] [in a new window]   Figure 1. Effect of 12/15-LO gene disruption on aortic sinus atherosclerosis in LDL-R-/- mice. 12/15-LO-/- and 12/15-LO+/+ LDL-R-/- mice were fed a Paigen diet for 3 and 9 weeks. At death, hearts were removed and sectioned at level of aortic sinus for evaluation of atherosclerotic lesion development after staining with oil red O (A and B). After high-fat diet for 9 weeks, aortas were removed, cleaned, and stained with Sudan IV as described in Methods for evaluation of aortic coverage (C). *P<0.01; **P<0.001; ***P<0.0001.

To further confirm the results of aortic root atherogenesis, we assessed the extent of atherosclerosis throughout the aortas by en face methodology. Because our previous observations indicated that after 3 weeks on a high-fat diet, no quantifiable lesions were observed throughout the aorta, lesions were evaluated after 9, 12, and 18 weeks of diet. Indeed, aortic surface lesion coverage was significantly reduced in LDL-R^-/-/12/15-LO^-/- mice at all time points versus LDL-R^-/-/12/15-LO^+/+ control mice.

Mice fed a Western-type high-fat diet were evaluated in an additional study for atherosclerotic lesion coverage of the aorta. Twelve as well as 18 weeks of feeding with a Western diet resulted in a significant attenuation in the extent of plaque formation in the double-knockout compared with LDL-R^-/- mice (Figure 2).

View larger version (34K): [in this window] [in a new window]   Figure 2. 12/15-LO gene disruption attenuates aortic lesion atherogenesis induced by 12 to 18 weeks of Western-type diet. 12/15-LO-/- and 12/15-LO+/+/LDL-R-/- mice (male and female) were fed a Western diet for 12 (n=13 to 16 mice/group) and 18 (n=11 to 12 mice/group) weeks. Extent of atherosclerosis was evaluated by calculating aortic surface coverage. *P<0.0001; **P=0.0004.

To investigate whether 12/15-LO gene disruption influenced plaque cellular composition in addition to plaque size, we made a quantitative assessment of macrophage and lymphocyte numbers. Early atherosclerotic lesions (after 3 weeks of Paigen diet) from LDL-R^-/-/12/15-LO^-/- mice showed a macrophage content (83.7±5.9%) similar to that in early lesions from LDL-R^-/-/12/15-LO^+/+ mice (87.3±1.5%; Figure 3). More advanced lesions (9 weeks of high-fat diet) from 12/15-LO^-/- mice were also found to exhibit a content of macrophages (82.5±5.5%) similar to that of control mice (73.5±6.6%). No differences were observed in the lymphocyte (CD3⁺) content between lesions of LDL-R^-/-/12/15-LO^-/- mice fed a Paigen diet for 3 weeks (3.0±1.1%) and control mice (2.6±1.0%; Figure 3) or 9 weeks (4.1±1.1% and 3.2±0.7%, respectively).

View larger version (20K): [in this window] [in a new window]   Figure 3. Plaque composition in LDL-R-/-/12/15-LO-/- mice. Frozen sections from LDL-/-/12/15-LO-/- and 12/15-LO+/+ mice fed a Paigen diet for 3 and 9 weeks were subjected to immunostaining with anti–Mac 1 antibodies (to evaluate macrophage content; A) or with anti-CD3 antibodies (to assess lymphocyte content; B). Results represent mean±SD of 3 sections from 5 to 6 mice in each group.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
In the present study, we address the role of 12/15-LO expression in atherogenesis. The optimal means of testing the hypothesis that this enzyme is proatherogenic is by crossbreeding experiments, identifying the effects of isolated gene disruptions. Indeed, Cyrus et al¹⁷ recently provided compelling evidence of the role of 12/15-LO in murine atherogenesis by crossing apoE-knockout with 12/15-LO^-/- mice, showing that early as well as advanced lesions were reduced. These observations are strongly supported by our recent observations in which overexpression of the human 15-LO gene in the vessel walls of LDL-R^-/- mice by use of the relatively endothelium-specific preproendothelin promotor enhanced early atherogenesis.¹⁴ Other than supporting the role of LO in atherogenesis, this study also indicated a relevant cellular compartment (ie, the endothelial cell) that could participate in LO-mediated oxidative modification culminating in accelerated atherosclerosis. These studies, however, are challenged by results from Shen et al¹⁸ showing that overexpression of 15-LO in rabbit macrophages results in suppression of plaque formation. The explanations that have been provided for the apparently conflicting results include the different animal models (rabbits versus mice) and the specific cellular target (macrophages) used by Shen et al.¹⁸

In the present study, we found that disruption of the 12/15-LO gene resulted in diminished atherosclerotic lesion formation induced by high-fat diet in LDL-R^-/- mice, consistent with the findings in apoE-knockout mice. It appeared that the differences in early lesion formation in our present study were relatively less pronounced than those obtained by Cyrus et al¹⁷ that explored spontaneous hypercholesterolemia in apoE-knockout mice. These differences may be explained by the use of a high-fat diet in our experiment that resulted in total cholesterol levels that significantly exceeded those obtained in the apoE^-/- study,¹⁷ thus possibly partially overwhelming the isolated effect of 12/15-LO–mediated LDL oxidation. As in the previous work,¹⁷ however, we also found that the 12/15-LO gene disruption did not appear to influence the lipid profile, thus excluding this factor as a possible explanation for the reduced lesion formation.

Several explanations have been provided for the mechanisms by which the cytosolic 12/15-LO enzyme could mediate extracellular LDL oxidation.²⁷ Hydroperoxides formed by LO could be transferred across the cell membrane, thereby contacting extracellular LDL. This is supported by the observation that incubation of LDL with fibroblasts transfected with 15-LO enhances generation of lipid hydroperoxides.^28–30 Alternatively, 12/15-HETE produced by local monocyte-macrophage activation could serve as ligand for peroxisome proliferator–activated receptor-, resulting in CD36 (a candidate scavenger receptor for oxLDL) upregulation with a consequent enhanced plaque formation.^31,32 Regardless of the mechanism, LO-generated products may also contribute to atherosclerosis by inducing a proinflammatory state by recruitment of macrophages and T lymphocytes and activating the cells already present within the lesion. Indeed, T-cell activation has been shown to occur within the atherosclerotic plaque,³³ and CD4-positive clone–producing T-helper type 1 cytokines (interferon-) have been isolated from human lesions.³⁴ To explore the relevance of this possibility in our model, we performed a quantitative analysis of the cellular components within the lesions. The results indicate that cellular composition was similar between LDL-R^-/-/12/15-LO^-/- and LDL-R^-/- mice, as reflected by the total macrophage and T-lymphocyte numbers within the lesions.

In conclusion, our results demonstrate that 12/15-LO gene disruption reduces atherosclerosis in LDL-R^-/- mice without influencing plaque composition. These data reinforce previous observations in the apoE mouse and indicate that 12/15-LO inhibition could represent an attractive treatment strategy for atherosclerosis.

The mice were fed a high-cholesterol, high-fat diet. Animals (n=10 to 11) from each group were bled at 0, 3, 6, and 9 weeks in 2 independent studies. Plasma cholesterol and triglyceride concentrations were determined as described in Methods. Results represent the mean±SEM.

	Acknowledgments

 
This study was supported in part by NIH grant HL-53558 (Dr Funk).

Received March 30, 2001; revision received June 7, 2001; accepted June 21, 2001.

	References

Top Abstract Introduction Methods Results Discussion References

 
1. Steinberg D, Parthasarathy S, Carew TE, et al. Beyond cholesterol: modifications of low-density lipoprotein that increase its atherogenicity. N Engl J Med. . 1989; 320: 915–924.[Medline] [Order article via Infotrieve]

2. Steinberg D. Oxidative modification of LDL and atherogenesis. The 1995 Lewis A. Conner Memorial Lecture. Circulation. . 1997; 95: 1062–1071.[Free Full Text]

3. Heinecke JW. Oxidants and antioxidants in the pathogenesis of atherosclerosis: implications for the oxidized low density lipoprotein hypothesis. Atherosclerosis. . 1998; 141: 1–15.[Medline] [Order article via Infotrieve]

4. Stephens, NG, Parsons A, Schofield PM, et al. Randomised controlled trial of vitamin E in patients with coronary disease: Cambridge Heart Antioxidant Study (CHAOS). Lancet. . 1996; 347: 781–786.[Medline] [Order article via Infotrieve]

5. Boaz M, Smetana S, Weinstein T, et al. Secondary prevention with antioxidants of cardiovascular disease in endstage renal disease (SPACE): randomized placebo controlled trial. Lancet. . 2000; 356: 1213–1218.[Medline] [Order article via Infotrieve]

6. Rapola JM, Virtamo J, Ripatti S, et al. Randomised trial of alpha-tocopherol and beta-carotene supplements on incidence of major coronary events in men with previous myocardial infraction. Lancet. . 1997; 349: 1715–1720.[Medline] [Order article via Infotrieve]

7. Dietary supplementation with n-3 polyunsaturated fatty acids and vitamin E after myocardial infarction: results of the GISSI-Prevenzione trial. Gruppo Italiano per lo Studio della Sopravvivenza nell’Infarto miocardico. Lancet. . 1999; 354: 447–455.[Medline] [Order article via Infotrieve]

8. Yusuf S, Dagenais G, Pogue J, et al. Vitamin E supplementation and cardiovascular events in high-risk patients. The Heart Outcomes Prevention Evaluation Study Investigators. N Engl J Med. . 2000; 342: 154–160.[Abstract/Free Full Text]

9. Funk CD. The molecular biology of mammalian lipoxygenases and the quest for eicosanoid functions using lipoxygenase-deficient mice. Biochim Biophys Acta. . 1996; 1304: 65–84.[Medline] [Order article via Infotrieve]

10. Belkner J, Stender H, Kuhn H. The rabbit 15-lipoxygenase preferentially oxygenates LDL cholesterol esters, and this reaction does not require vitamin E. J Biol Chem. . 1998; 273: 23225–23232.[Abstract/Free Full Text]

11. Sloane DL, Leung R, Craik CS, et al. A primary determinant for lipoxygenase positional specificity. Nature. . 1991; 354: 149–152.[Medline] [Order article via Infotrieve]

12. Yla-Herttuala S, Rosenfeld ME, Parthasarathy S, et al. Colocalization of 15-lipoxygenase mRNA and protein with epitopes of oxidized low density lipoprotein in macrophage-rich areas of atherosclerotic lesions. Proc Natl Acad Sci U S A. . 1990; 87: 6959–6963.[Abstract/Free Full Text]

13. Yla-Herttuala S, Luoma J, Vilta H, et al. Transfer of 15-lipoxygenase gene into rabbit iliac arteries results in the appearance of oxidation-specific lipid-protein adducts characteristic of oxidized low density lipoprotein. J Clin Invest. . 1995; 95: 2692–2698.

14. Harats D, Shaish A, George J, et al. Overexpression of 15-lipoxygenase in vascular endothelium accelerates early atherogenesis in LDL receptor–deficient mice. Arterioscler Thromb Vasc Biol. . 2000; 20: 2100–2105.[Abstract/Free Full Text]

15. Sendobry SM, Cornicelli JA, Welch K, et al. Attenuation of diet-induced atherosclerosis in rabbits with a highly selective 15-lipoxygenase inhibitor lacking significant antioxidant properties. Br J Pharmacol. . 1997; 120: 1199–1206.[Medline] [Order article via Infotrieve]

16. Bocan T, Rosebury WS, Mueller SB, et al. A specific 15-lipoxygenase inhibitor limits the progression and monocyte-macrophage enrichment of hypercholesterolemia-induced atherosclerosis in the rabbit. Atherosclerosis. . 1998; 136: 203–216.[Medline] [Order article via Infotrieve]

17. Cyrus T, Witztum JL, Rader DJ, et al. Disruption of the 12/15-lipoxygenase gene diminishes atherosclerosis in apo E–deficient mice. J Clin Invest. . 1999; 103: 1597–1604.[Medline] [Order article via Infotrieve]

18. Shen J, Herderick E, Cornhill JF, et al. Macrophage-mediated 15-lipoxygenase expression protects against atherosclerosis development. J Clin Invest. . 1997; 98: 2201–2208.[Medline] [Order article via Infotrieve]

19. Kuhn H, Chan L. The role of 15-lipoxygenase in atherogenesis: pro- and antiatherogenic actions. Curr Opin Lipidol. . 1997; 8: 111–117.[Medline] [Order article via Infotrieve]

20. Sun D, Funk CD. Disruption of 12/15-lipoxygenase expression in peritoneal macrophages: enhanced utilization of the 5-lipoxygenase pathway and diminished oxidation of low density lipoprotein. J Biol Chem. . 1996; 271: 24055–24062.[Abstract/Free Full Text]

21. Ishibashi S, Brown MN, Goldstein JL, et al. Hypercholesterolemia in LDL receptor knockout mice and its reversal by adenovirus mediated gene delivery. J Clin Invest. . 1993; 92: 883–893.

22. Esterbauer H, Striegl G, Puhl H, et al. Continuous monitoring of in vitro oxidation of human low density lipoprotein. Free Radic Res Commun. . 1983; 6: 67–75.

23. Paigen B, Morrow A, Holmes PA, et al. Quantitative assessment of atherosclerotic lesions in mice. Atherosclerosis. . 1987; 68: 231–240.[Medline] [Order article via Infotrieve]

24. Shaish A, George J, Gilburd B, et al. Dietary ß-carotene and -tocopherol combination does not inhibit atherogenesis in an apoE-deficient mouse model. Arterioscler Thromb Vasc Biol. . 1999; 19: 1470–1475.[Abstract/Free Full Text]

25. Palinski W, Ord VA, Plump AS, et al. ApoE-deficient mice are a model of lipoprotein oxidation in atherogenesis: demonstration of oxidation-specific epitopes in lesions and high titers of autoantibodies to malondialdehyde-lysine in serum. Arterioscler Thromb. . 1994; 14: 605–616.[Abstract/Free Full Text]

26. George J, Shoenfeld Y, Gilburd B, et al. Requisite role for IL-4 in the enhancement of fatty streaks by immunization with heat shock protein 65. Circ Res. . 2000; 86: 1203–1210.[Abstract/Free Full Text]

27. Heinecke JW. Mechanisms of oxidative damage of low density lipoprotein in human atherosclerosis. Curr Opin Lipidol. . 1997; 8: 268–274.[Medline] [Order article via Infotrieve]

28. Benz DJ, Mol M, Ezaki M. et al. Enhanced levels of lipoperoxides in low density lipoprotein incubated with murine fibroblast expressing high levels of human 15-lipoxygenase. J Biol Chem. . 1995; 270: 5191–5197.[Abstract/Free Full Text]

29. Ezaki M, Witztum JL, Steinberg D. Lipoperoxides in LDL incubated with fibroblasts that overexpress 15-lipoxygenase. J Lipid Res. . 1995; 36: 1996–2004.[Abstract]

30. Sigari F, Lee C, Witztum JL, et al. Fibroblasts that overexpress 15-lipoxygenase generate bioactive and minimally modified LDL. Arterioscler Thromb Vasc Biol. . 1997; 17: 3639–3645.[Abstract/Free Full Text]

31. Tontonoz P, Nagy L, Alvarez JG, et al. PPAR gamma promotes monocyte/macrophage differentiation and uptake of oxidized LDL. Cell. . 1998; 93: 241–252.[Medline] [Order article via Infotrieve]

32. Nozaki S, Kashiwagi H, Yamashita S, et al. Reduced uptake of oxidized low density lipoproteins in monocyte-derived macrophages from CD36-deficient subjects. J Clin Invest. . 1995; 96: 1859–1865.

33. Hansson GK, Holm J, Jonasson L. Detection of activated T lymphocytes in the human atherosclerotic plaque. Am J Pathol. . 1989; 135: 169–175.[Abstract]

34. Stemme S, Faber B, Holm J. T lymphocytes from human atherosclerotic plaques recognize oxidized low density lipoprotein. Proc Natl Acad Sci U S A. . 1995; 92: 3893–3897.[Abstract/Free Full Text]

This article has been cited by other articles:

				Y. Kayama, T. Minamino, H. Toko, M. Sakamoto, I. Shimizu, H. Takahashi, S. Okada, K. Tateno, J. Moriya, M. Yokoyama, et al. Cardiac 12/15 lipoxygenase-induced inflammation is involved in heart failure J. Exp. Med., July 6, 2009; 206(7): 1565 - 1574. [Abstract] [Full Text] [PDF]

				S.-H. Choi, R. Harkewicz, J. H. Lee, A. Boullier, F. Almazan, A. C. Li, J. L. Witztum, Y. S. Bae, and Y. I. Miller Lipoprotein Accumulation in Macrophages via Toll-Like Receptor-4-Dependent Fluid Phase Uptake Circ. Res., June 19, 2009; 104(12): 1355 - 1363. [Abstract] [Full Text] [PDF]

				D. Steinberg The LDL modification hypothesis of atherogenesis: an update J. Lipid Res., April 1, 2009; 50(Supplement): S376 - S381. [Abstract] [Full Text] [PDF]

				A. J. Merched, K. Ko, K. H. Gotlinger, C. N. Serhan, and L. Chan Atherosclerosis: evidence for impairment of resolution of vascular inflammation governed by specific lipid mediators FASEB J, October 1, 2008; 22(10): 3595 - 3606. [Abstract] [Full Text] [PDF]

				A. P. Lillis, L. B. Van Duyn, J. E. Murphy-Ullrich, and D. K. Strickland LDL Receptor-Related Protein 1: Unique Tissue-Specific Functions Revealed by Selective Gene Knockout Studies Physiol Rev, July 1, 2008; 88(3): 887 - 918. [Abstract] [Full Text] [PDF]

				R. Harkewicz, K. Hartvigsen, F. Almazan, E. A. Dennis, J. L. Witztum, and Y. I. Miller Cholesteryl Ester Hydroperoxides Are Biologically Active Components of Minimally Oxidized Low Density Lipoprotein J. Biol. Chem., April 18, 2008; 283(16): 10241 - 10251. [Abstract] [Full Text] [PDF]

				M. McDuffie, N. A. Maybee, S. R. Keller, B. K. Stevens, J. C. Garmey, M. A. Morris, E. Kropf, C. Rival, K. Ma, J. D. Carter, et al. Nonobese Diabetic (NOD) Mice Congenic for a Targeted Deletion of 12/15-Lipoxygenase Are Protected From Autoimmune Diabetes Diabetes, January 1, 2008; 57(1): 199 - 208. [Abstract] [Full Text] [PDF]

				A. Mor, D. Planer, G. Luboshits, A. Afek, S. Metzger, T. Chajek-Shaul, G. Keren, and J. George Role of Naturally Occurring CD4+CD25+ Regulatory T Cells in Experimental Atherosclerosis Arterioscler. Thromb. Vasc. Biol., April 1, 2007; 27(4): 893 - 900. [Abstract] [Full Text] [PDF]

				C. D. Funk Lipoxygenase Pathways as Mediators of Early Inflammatory Events in Atherosclerosis. Arterioscler. Thromb. Vasc. Biol., June 1, 2006; 26(6): 1204 - 1206. [Full Text] [PDF]

				D. T. Bolick, S. Srinivasan, A. Whetzel, L. C. Fuller, and C. C. Hedrick 12/15 Lipoxygenase Mediates Monocyte Adhesion to Aortic Endothelium in Apolipoprotein E-Deficient Mice Through Activation of RhoA and NF-{kappa}B Arterioscler. Thromb. Vasc. Biol., June 1, 2006; 26(6): 1260 - 1266. [Abstract] [Full Text] [PDF]

				J. Huber, A. Furnkranz, V. N. Bochkov, M. K. Patricia, H. Lee, C. C. Hedrick, J. A. Berliner, B. R. Binder, and N. Leitinger Specific monocyte adhesion to endothelial cells induced by oxidized phospholipids involves activation of cPLA2 and lipoxygenase J. Lipid Res., May 1, 2006; 47(5): 1054 - 1062. [Abstract] [Full Text] [PDF]

				A. Boullier, Y. Li, O. Quehenberger, W. Palinski, I. Tabas, J. L. Witztum, and Y. I. Miller Minimally Oxidized LDL Offsets the Apoptotic Effects of Extensively Oxidized LDL and Free Cholesterol in Macrophages Arterioscler. Thromb. Vasc. Biol., May 1, 2006; 26(5): 1169 - 1176. [Abstract] [Full Text] [PDF]

				S. Xu, B. Jiang, K. A. Maitland, H. Bayat, J. Gu, J. L. Nadler, S. Corda, G. Lavielle, T. J. Verbeuren, A. Zuccollo, et al. The Thromboxane Receptor Antagonist S18886 Attenuates Renal Oxidant Stress and Proteinuria in Diabetic Apolipoprotein E-Deficient Mice Diabetes, January 1, 2006; 55(1): 110 - 119. [Abstract] [Full Text] [PDF]

				D. T. Bolick, A. W. Orr, A. Whetzel, S. Srinivasan, M. E. Hatley, M. A. Schwartz, and C. C. Hedrick 12/15-Lipoxygenase Regulates Intercellular Adhesion Molecule-1 Expression and Monocyte Adhesion to Endothelium Through Activation of RhoA and Nuclear Factor-{kappa}B Arterioscler. Thromb. Vasc. Biol., November 1, 2005; 25(11): 2301 - 2307. [Abstract] [Full Text] [PDF]

				A. M. Taylor, R. Hanchett, R. Natarajan, C. C. Hedrick, S. Forrest, J. L. Nadler, and C. A. McNamara The Effects of Leukocyte-Type 12/15-Lipoxygenase on Id3-Mediated Vascular Smooth Muscle Cell Growth Arterioscler. Thromb. Vasc. Biol., October 1, 2005; 25(10): 2069 - 2074. [Abstract] [Full Text] [PDF]

				Y. I. Miller, S. Viriyakosol, D. S. Worrall, A. Boullier, S. Butler, and J. L. Witztum Toll-Like Receptor 4-Dependent and -Independent Cytokine Secretion Induced by Minimally Oxidized Low-Density Lipoprotein in Macrophages Arterioscler. Thromb. Vasc. Biol., June 1, 2005; 25(6): 1213 - 1219. [Abstract] [Full Text] [PDF]

				J. Belkner, P. Chaitidis, H. Stender, C. Gerth, R. J. Kuban, T. Yoshimoto, and H. Kuhn Expression of 12/15-Lipoxygenase Attenuates Intracellular Lipid Deposition During In Vitro Foam Cell Formation Arterioscler. Thromb. Vasc. Biol., April 1, 2005; 25(4): 797 - 802. [Abstract] [Full Text] [PDF]

				P. B. Anning, B. Coles, A. Bermudez-Fajardo, P. E.M. Martin, B. S. Levison, S. L. Hazen, C. D. Funk, H. Kuhn, and V. B. O'Donnell Elevated Endothelial Nitric Oxide Bioactivity and Resistance to Angiotensin-Dependent Hypertension in 12/15-Lipoxygenase Knockout Mice Am. J. Pathol., March 1, 2005; 166(3): 653 - 662. [Abstract] [Full Text] [PDF]

				E. K. Rydberg, A. Krettek, C. Ullstrom, K. Ekstrom, P.-A. Svensson, L. M.S. Carlsson, A.-C. Jonsson-Rylander, G. I. Hansson, W. McPheat, O. Wiklund, et al. Hypoxia Increases LDL Oxidation and Expression of 15-Lipoxygenase-2 in Human Macrophages Arterioscler. Thromb. Vasc. Biol., November 1, 2004; 24(11): 2040 - 2045. [Abstract] [Full Text] [PDF]

				P. J. Barter, S. Nicholls, K.-A. Rye, G.M. Anantharamaiah, M. Navab, and A. M. Fogelman Antiinflammatory Properties of HDL Circ. Res., October 15, 2004; 95(8): 764 - 772. [Abstract] [Full Text] [PDF]

				Y. Huo, L. Zhao, M. C. Hyman, P. Shashkin, B. L. Harry, T. Burcin, S. B. Forlow, M. A. Stark, D. F. Smith, S. Clarke, et al. Critical Role of Macrophage 12/15-Lipoxygenase for Atherosclerosis in Apolipoprotein E-Deficient Mice Circulation, October 5, 2004; 110(14): 2024 - 2031. [Abstract] [Full Text] [PDF]

				R. Stocker and J. F. Keaney Jr. Role of Oxidative Modifications in Atherosclerosis Physiol Rev, October 1, 2004; 84(4): 1381 - 1478. [Abstract] [Full Text] [PDF]

				R. Natarajan and J. L. Nadler Lipid Inflammatory Mediators in Diabetic Vascular Disease Arterioscler. Thromb. Vasc. Biol., September 1, 2004; 24(9): 1542 - 1548. [Abstract] [Full Text] [PDF]

				H. Kuhn Lipoxygenases in the Cardiovascular System Circ. Res., June 25, 2004; 94(12): 1527 - 1529. [Full Text] [PDF]

				M. Navab, G. M. Ananthramaiah, S. T. Reddy, B. J. Van Lenten, B. J. Ansell, G. C. Fonarow, K. Vahabzadeh, S. Hama, G. Hough, N. Kamranpour, et al. Thematic review series: The Pathogenesis of Atherosclerosis The oxidation hypothesis of atherogenesis: the role of oxidized phospholipids and HDL J. Lipid Res., June 1, 2004; 45(6): 993 - 1007. [Abstract] [Full Text] [PDF]

				K. B. Reilly, S. Srinivasan, M. E. Hatley, M. K. Patricia, J. Lannigan, D. T. Bolick, G. Vandenhoff, H. Pei, R. Natarajan, J. L. Nadler, et al. 12/15-Lipoxygenase Activity Mediates Inflammatory Monocyte/Endothelial Interactions and Atherosclerosis in Vivo J. Biol. Chem., March 5, 2004; 279(10): 9440 - 9450. [Abstract] [Full Text] [PDF]

				P. A. VanderLaan, C. A. Reardon, and G. S. Getz Site Specificity of Atherosclerosis: Site-Selective Responses to Atherosclerotic Modulators Arterioscler. Thromb. Vasc. Biol., January 1, 2004; 24(1): 12 - 22. [Abstract] [Full Text] [PDF]

				X. Tang, N. Spitzbarth, H. Kuhn, P. Chaitidis, and W. B. Campbell Interleukin-13 Upregulates Vasodilatory 15-Lipoxygenase Eicosanoids in Rabbit Aorta Arterioscler. Thromb. Vasc. Biol., October 1, 2003; 23(10): 1768 - 1774. [Abstract] [Full Text] [PDF]

				Y. I. Miller, D. S. Worrall, C. D. Funk, J. R. Feramisco, and J. L. Witztum Actin Polymerization in Macrophages in Response to Oxidized LDL and Apoptotic Cells: Role of 12/15-Lipoxygenase and Phosphoinositide 3-Kinase Mol. Biol. Cell, October 1, 2003; 14(10): 4196 - 4206. [Abstract] [Full Text] [PDF]

				M. E. Hatley, S. Srinivasan, K. B. Reilly, D. T. Bolick, and C. C. Hedrick Increased Production of 12/15 Lipoxygenase Eicosanoids Accelerates Monocyte/Endothelial Interactions in Diabetic db/db Mice J. Biol. Chem., July 3, 2003; 278(28): 25369 - 25375. [Abstract] [Full Text] [PDF]

				H. Kuhn, M. Anton, C. Gerth, and A. Habenicht Amino Acid Differences in the Deduced 5-Lipoxygenase Sequence of CAST Atherosclerosis-Resistance Mice Confer Impaired Activity When Introduced Into the Human Ortholog Arterioscler. Thromb. Vasc. Biol., June 1, 2003; 23(6): 1072 - 1076. [Abstract] [Full Text] [PDF]

				M. A. Reddy, Y.-S. Kim, L. Lanting, and R. Natarajan Reduced Growth Factor Responses in Vascular Smooth Muscle Cells Derived from 12/15-Lipoxygenase-Deficient Mice Hypertension, June 1, 2003; 41(6): 1294 - 1300. [Abstract] [Full Text] [PDF]

				R. Spanbroek, R. Grabner, K. Lotzer, M. Hildner, A. Urbach, K. Ruhling, M. P. W. Moos, B. Kaiser, T. U. Cohnert, T. Wahlers, et al. Expanding expression of the 5-lipoxygenase pathway within the arterial wall during human atherogenesis PNAS, February 4, 2003; 100(3): 1238 - 1243. [Abstract] [Full Text] [PDF]

				L. Fuentes, M. Hernandez, F. J. Fernandez-Aviles, M. S. Crespo, and M. L. Nieto Cooperation Between Secretory Phospholipase A2 and TNF-Receptor Superfamily Signaling: Implications for the Inflammatory Response in Atherogenesis Circ. Res., October 18, 2002; 91(8): 681 - 688. [Abstract] [Full Text] [PDF]

				L. Zhao, C. A. Cuff, E. Moss, U. Wille, T. Cyrus, E. A. Klein, D. Pratico, D. J. Rader, C. A. Hunter, E. Pure, et al. Selective Interleukin-12 Synthesis Defect in 12/15-Lipoxygenase-deficient Macrophages Associated with Reduced Atherosclerosis in a Mouse Model of Familial Hypercholesterolemia J. Biol. Chem., September 13, 2002; 277(38): 35350 - 35356. [Abstract] [Full Text] [PDF]

				D. A. Wong, Y. Kita, N. Uozumi, and T. Shimizu Discrete Role for Cytosolic Phospholipase A2{alpha} in Platelets: Studies Using Single and Double Mutant Mice of Cytosolic and Group IIA Secretory Phospholipase A2 J. Exp. Med., August 5, 2002; 196(3): 349 - 357. [Abstract] [Full Text] [PDF]

				M. E. Rosenfeld Leukocyte Recruitment Into Developing Atherosclerotic Lesions: The Complex Interaction Between Multiple Molecules Keeps Getting More Complex Arterioscler. Thromb. Vasc. Biol., March 1, 2002; 22(3): 361 - 363. [Full Text] [PDF]

This Article Abstract Full Text (PDF) Alert me when this article is cited 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 George, J. Articles by Harats, D. Search for Related Content PubMed PubMed Citation Articles by George, J. Articles by Harats, D. Related Collections Pathophysiology