Disruption of the 12/15-lipoxygenase gene diminishes atherosclerosis in apo E-deficient mice

Abstract

Atherosclerosis may be viewed as an inflammatory disease process that includes early oxidative modification of LDLs, leading to foam cell formation. This "oxidation hypothesis" has gained general acceptance in recent years, and evidence for the role of lipoxygenases in initiation of, or participation in, the oxidative process is accumulating. However, the relative contribution of macrophage-expressed lipoxygenases to atherogenesis in vivo remains unknown. Here, we provide in vivo evidence for the role of 12/15-lipoxygenase in atherogenesis and demonstrate diminished plasma IgG autoantibodies to oxidized LDL epitopes in 12/15-lipoxygenase knockout mice crossbred with atherosclerosis-prone apo E-deficient mice (apo E-/-/L-12LO-/-). In chow-fed 15-week-old apo E-/-/L-12LO-/- mice, the extent of lesions in whole-aorta en face preparations (198 +/- 60 microm2) was strongly reduced (P < 0.001, n = 12) when compared with 12/15-lipoxygenase-expressing controls (apo E-/-/L-12LO+/+), which showed areas of lipid deposition (15,700 +/- 2,688 microm2) in the lesser curvature of the aortic arch, branch points, and in the abdominal aorta. These results were observed despite cholesterol, triglyceride, and lipoprotein levels that were similar to those in apo E-deficient mice. Evidence for reduced lesion development was observed even at 1 year of age in apo E-/-/L-12LO-/- mice. The combined data indicate a role for 12/15-lipoxygenase in the pathogenesis of atherosclerosis and suggest that inhibition of this enzyme may decrease disease progression.

Figure 1

Lipoprotein profiles in apo E–deficient mice are not altered by 12/15-lipoxygenase deficiency. Representative plasma lipoprotein profiles of apo E^–/–/L-12LO^–/– double-knockout mice and apo E^–/–/L-12LO^+/– control mice. Pooled plasmas from two or three 15-week-old mice on chow diet (n = 2–4) were fractionated by FPLC, using 2 Superose 6 columns in series. Fractions were then assayed for cholesterol in duplicates. All values are expressed as micrograms per fraction per 100 μL of plasma.

Figure 2

The ability of 12/15-lipoxygenase–deficient macrophages to synthesize the arachidonate metabolites 12-HETE/15-HETE is abolished. Profiles of monohydroxy arachidonic acid metabolites synthesized by apo E^–/–/L-12LO^+/+ macrophages (left), apo E^–/–/L-12LO^+/– macrophages (middle), and apo E^–/–/L-12LO^–/– macrophages (right) incubated with arachidonic acid and analyzed by RP-HPLC. 12-HETE production in the left panel was 0.63 nmol/10⁶ cells.

Figure 3

12/15-lipoxygenase deficiency results in diminished fatty lesions in apo E-deficient mice. (a) Representative Sudan IV–stained aortas of an apo E^–/–/L-12LO^–/– double-knockout mouse (left) and an apo E knockout mouse heterozygous for L-12LO (right). Mice were raised on a normal chow diet and sacrificed at 15 weeks of age (n = 12). Atheromatous plaques have already developed in the lesser curvature of the aortic arch (asterisk), at the ostia of the left common carotid artery, and the left subclavian artery (arrow), as well as in the abdominal aorta between the ostia of the left and right renal artery (double asterisk), whereas the aorta of the double-knockout mouse remains free of any lesion development at this age. (b and c) Atherosclerotic average lesion area in en face preparations (n = 12 per group) (b) and aortic sinus areas (n = 12 for L-12LO^+/– and L-12LO^–/–; n = 9 for L-12LO^+/+) (c) in apo E^–/– mice lacking L-12LO^–/–, expressing 1 allele of L-12LO (L-12LO^+/–), or expressing both alleles (L-12LO^+/+). Mice fed a normal chow diet were sacrificed at 15 weeks of age. Values are mean ± SEM. **P < 0.0001 (t test). For comparison between groups in c: *P = 0.015 for L-12LO^+/+ vs. L-12LO^–/– (Mann-Whitney test), P = 0.10 for L-12LO^+/– vs. L-12LO^–/– (Mann-Whitney test), and P = 0.60 for L-12LO^+/+ vs. L-12LO^+/– (t test).

Figure 4

Analysis of lesions and L-12LO protein in aortic sinus sections and macrophages. (a–g) Aortic sinus sections from an apo E^–/–/L-12LO^+/– mouse (a, c, e, and g; total lesion area = 60,930 μm²) and an apo E^–/–/L-12LO^–/– double-knockout mouse (b, d, and f; total lesion area = 16,129 μm²) were stained for lipid lesions (oil red O) (a and b) and immunostained for macrophage content (MOMA-2 antibody) (c and d) and for 12/15-lipoxygenase (polyclonal antibody against L-12LO) (e–g). The image in g is magnified from the upper-right lesion in e to show foamy macrophage composition. Sections are not adjacent but are within the first 300 μm of the proximal aorta, starting at the aortic sinus. Peritoneal macrophages were also immunostained with antibody against L-12LO (h and i). Macrophages from a double-knockout animal (i) do not express 12-lipoxygenase, and about 40% of the macrophages from an apo E^–/–/L-12LO^+/– mouse (h) express L-12LO, which is consistent with previous in situ hybridization data (45). More than 98% of the cells in this figure are macrophages.

Figure 5

12/15-lipoxygenase deficiency delays progression of lesion development in apo E–deficient mice. (a) Representative Sudan IV–stained aortas of an apo E^–/–/L-12LO^–/– double-knockout mouse (left) and an apo E knockout mouse heterozygous for L-12LO (right). Mice were raised on a normal chow diet and sacrificed at 1 year of age (n = 12 and n = 10, respectively). Atheromatous plaques are at a much more advanced stage in the apo E^–/–/L-12LO^+/– group and include large parts of the thoracic and abdominal aorta, with near occlusion in the renal artery area. (b) Atherosclerotic average lesion area in en face preparations at 1 year of age. Values are mean ± SEM. **P < 0.0001 (Mann-Whitney test).

Figure 6

Diminished titers of antibodies directed toward oxLDL epitopes in apo E^–/– L-12LO^–/– mice. Solid-phase chemiluminescent immunoassays of autoantibodies to oxLDL and MDA-LDL epitopes in plasma samples of apo E^–/–/L-12LO^+/– and apo E^–/–/L-12LO^–/– mice (n = 9 and n = 15, respectively; mean ± SEM). *P = 0.008 for autoantibody titers to MDA-LDL, and P = 0.03 for autoantibody titers to oxLDL (t test). Data are expressed as relative light units per 100 milliseconds (RLU/100 ms).