Identification of 5-Lipoxygenase as a Major Gene Contributing to Atherosclerosis Susceptibility in Mice

Abstract

Abstract-We previously reported the identification of a locus on mouse chromosome 6 that confers almost total resistance to atherogenesis, even on a hypercholesterolemic (LDL receptor-null) background. 5-Lipoxygenase (5-LO) is the rate-limiting enzyme in leukotriene synthesis and was among the chromosome 6 locus candidate genes that we examined. The levels of 5-LO mRNA were reduced about 5-fold in a congenic strain, designated CON6, containing the resistant chromosome 6 region derived from the CAST/Ei strain (CAST), as compared with the background C57BL/6J (B6) strain. 5-LO protein levels were similarly reduced in the CON6 mice. Sequencing of the 5-LO cDNA revealed several differences between CON6 and the B6 strain. To test the whether 5-LO is responsible for the resistant phenotype, we bred a 5-LO knockout allele onto an LDL receptor-null (LDLR Ϫ/Ϫ ) background. On this background, the mice bred poorly and only heterozygous 5-LO knockout mice were obtained. These mice showed a dramatic decrease (Ͼ26-fold; PϽ0.0005) in aortic lesion development, similar to the CON6 mice. Immunohistochemistry revealed that 5-LO was abundantly expressed in atherosclerotic lesions of apoE Ϫ/Ϫ and LDLR Ϫ/Ϫ deficient mice, appearing to colocalize with a subset of macrophages but not with all macrophage-staining regions. When bone marrow from 5-LO ϩ/Ϫ mice was transplanted into LDLR Ϫ/Ϫ , there was a significant reduction in atherogenesis, suggesting that macrophage 5-LO is responsible, at least in part, for the effect on atherosclerosis. These results indicate that 5-LO contributes importantly to the atherogenic process and they provide strong presumptive evidence that reduced 5-LO expression is partly responsible for the resistance to atherosclerosis in CON6 mice. This signals a cascade of leukocyte recruitment, further lipoprotein oxidation, foam cell formation, necrosis, and fibroproliferation. To identify genes that contribute to this complex process, we previously constructed a cross between an atherosclerosis-resistant mouse strain, CAST, and a susceptible strain, B6. A major locus for atherosclerosis was identified on mouse chromosome 6 and was subsequently confirmed with the congenic strain designated CON6 in which the central region of chromosome 6 from CAST was bred onto a B6 background. 3 These CON6 mice had reduced insulin levels and dramatically decreased lesion formation when bred onto an LDL receptornull (LDLR Ϫ/Ϫ ) background and fed an atherogenic diet. Moreover, bone marrow transplantation studies indicated that the resistant phenotype was conferred in part by bone marrowderived cells.In examining the congenic region for potential positional candidate genes, we observed that 5-lipoxygenase (5-LO) mapped directly underneath the linkage peak for the locus. 5-LO is the rate-limiting enzyme in leukotriene (LT) biosynthesis 4 and is expressed primarily in leukocytes, including monocytes and macrophages. 5 Leukotrienes are potent proinflammatory lipid mediators derived from arachidonic acid and have been shown to affect s...

The gene encoding 5-lipoxygenase activating protein confers risk of myocardial infarction and stroke

“…A 5-LO promoter variant is associated with abnormal carotid artery intima-media thickness and heightened inflammatory biomarkers 30 . In addition, antagonists of LTB4 block the development of atherosclerosis in apo-E-deficient and LDRLdeficient mice 31 , and a congenic mouse strain with a heterozygous deficiency of 5-LO shows resistance to atherosclerosis 16 , further supporting the idea that greater activity of the 5-LO pathway has a role in predisposition to atherosclerosis.…”

“…FLAP, together with 5-lipoxygenase (5-LO), is a regulator of the leukotriene biosynthetic pathway that has recently been implicated in the pathogenesis of atherosclerosis [16][17][18] . Therefore, ALOX5AP was a good candidate for the gene underlying the association with myocardial infarction.…”

“…Through suggestive linkage to a locus on chromosome 13q12-13, we identified the gene (ALOX5AP) encoding FLAP and found that a four-SNP haplotype in the gene confers a nearly two times greater risk of myocardial infarction and stroke. FLAP is a regulator 15 of a crucial pathway in the genesis of leukotriene inflammatory mediators, which are implicated in atherosclerosis both in a mouse model 16 and in human studies 17,18 . Males had the strongest association to the at-risk haplotype, and male carriers of the at-risk haplotype also had significantly greater production of leukotriene-B4 (LTB4), supporting the idea that proinflammatory activity has a role in the pathogenesis of myocardial infarction.…”

Identification of ALOX5 as a gene regulating adiposity and pancreatic function

“…In previous studies, we reported that 5-LO deficiency dramatically decreases aortic lesion development in two mouse models [3,4] and that humans carrying certain ALOX5 gene variants have increased carotid intima-media thickness [5]. Moreover, recent studies in humans and mice have demonstrated that various pharmacological modifiers of the 5-LO pathway decrease inflammatory biomarkers and aortic lesion formation, respectively [9,13,37], raising the possibility of developing 5-LO pathway inhibitors for the treatment of CVD.…”

“…Importantly, multiple lines of evidence support this concept, including biochemical, genetic and pharmacological studies in both mice and humans [2]. For example, we demonstrated that deficiency of the gene encoding 5-lipoxygenase (Alox5) in mice protects against the development of aortic lesions [3,4], and that promoter variants of the human ALOX5 gene are associated with increased carotid intima-media thickness [5]. Other groups have also reported findings that support the involvement of the 5-LO/LT pathway in CVD traits [6][7][8][9][10][11][12][13].…”