Research ArticleHEALTH AND MEDICINE

Targeting macrophage necroptosis for therapeutic and diagnostic interventions in atherosclerosis

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Science Advances  22 Jul 2016:
Vol. 2, no. 7, e1600224
DOI: 10.1126/sciadv.1600224
  • Fig. 1 Up-regulation of necroptotic genes in unstable atherosclerosis.

    (A) mRNA expression of RIP3 and MLKL in carotid endarterectomies (plaque) or macroscopically disease-free control arteries (normal). (B) RIP3 and MLKL mRNA expression in plaque samples from (A) classified as asymptomatic (stable) or symptomatic patients (unstable) [from the Biobank of Karolinska Endarterectomies (BiKE)]. *P ≤ 0.05, **P < 0.01, ****P < 0.0001 by Student’s t test. (C) Immunohistochemical analysis of pMLKL in human coronary arteries with early lesions with pathologic intimal thickening (n = 5 arterial segments) and advanced fibroatheroma lesions (n = 11 arterial segments). The graph depicts quantification of the pMLKL-positive area. *P ≤ 0.05. H&E, hematoxylin and eosin.

  • Fig. 2 OxLDL induces necroptotic cell death in macrophages.

    (A) BMDMs were treated with oxLDL (100 μg/ml) ± zVAD.fmk ± Nec-1 for 24 hours, and LDH (lactate dehydrogenase) release in the medium was measured. Data represent means ± SEM of five independent experiments. (B) Cell death in response to oxLDL ± zVAD.fmk in BMDMs from wild-type (WT) and Rip3−/− mice. (C) Western blot analysis of RIP3 after treatment with oxLDL ± zVAD.fmk ± Nec-1 for 8 hours. Band shift indicates phospho-RIP3 (pRIP3). (D) Western blot analysis of pMLKL after treatment with oxLDL for 12 hours or oxLDL ± zVAD.fmk for 8 hours. (E) Electron microscopy ultrastructural analysis of control and oxLDL-treated macrophages. Control macrophages had normal-looking cytoplasm, whereas oxLDL-treated macrophages had electron-light zones (arrows) that were not observed in control macrophages. Scale bar, 500 nm. (F) BMDMs were treated for 24 hours with medium alone (control) or medium containing 5 μM staurosporine (STS) (to induce apoptosis), oxLDL + zVAD, or LPS + zVAD (to induce necroptosis). Dead cells were collected, counted, resuspended in control medium, and applied to naïve BMDMs at a ratio of 3:1. After 2 hours, cells were washed six times with cold phosphate-buffered saline (PBS), and fluorescence intensity was quantified using a plate reader. The graph depicts a representative experiment with a mean ± SD of n = 4 experimental replicates repeated at least three times. ****P < 0.0001, ***P < 0.001 versus nontreated (control). (G) BMDMs were subjected to freeze-thaw to generate necrotic DAMPs, which were added to cells with or without oxLDL ± Nec-1 for 24 hours, and cell death was measured. (H) Western blot analysis of RIP3 after treatment with oxLDL ± zVAD.fmk ± Nec-1 with or without DAMPs for 8 hours. Band shift indicates phospho-RIP3. (I) Cell death in response to oxLDL ± zVAD.fmk ± necrotic freeze-thaw DAMPs in BMDMs from WT and Rip3−/− mice. (J) Cell death in BMDMs from WT and Casp1−/− mice in response to oxLDL ± zVAD.fmk for 24 hours. *P ≤ 0.05, **P < 0.01, ***P < 0.001 by one-way or two-way analysis of variance (ANOVA). ns, not significant.

  • Fig. 3 OxLDL induces the expression of RIP3 and MLKL.

    (A and B) BMDMs treated with oxLDL (100 μg/ml) ± zVAD.fmk for 3, 6, 12, or 24 hours were analyzed for gene expression of RIP3 (A) and MLKL (B) by qPCR (quantitative real-time polymerase chain reaction) and compared to control-treated cells. (C and D) Western blot analysis of RIP3 (C) and MLKL (D) expression in BMDMs treated with oxLDL. Quantification below of at least three independent experiments. (E) BMDMs were preincubated with 50 μM DPI and then treated with oxLDL or oxLDL + zVAD.fmk for 3 hours before measuring ROS levels. The graph shows mean ± SD of technical triplicates and is representative of at least three experiments. Statistical analysis was performed using two-way ANOVA. ***P < 0.001. (F) RIP3 or MLKL mRNA expression measured by qPCR in BMDMs treated with oxLDL ± zVAD.fmk, in the presence or absence of pretreatment with 50 μM DPI. (G and H) RAW macrophages were transfected with RIP3-promoter (G) or MLKL-promoter (H) luciferase constructs and treated for 6 or 24 hours with oxLDL in the presence or absence of pretreatment with 50 μM DPI before luciferase expression was examined and expressed as promoter-luciferase activation normalized to control (no treatment). *P ≤ 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 by one-way ANOVA.

  • Fig. 4 Radiolabeled Nec-1 can be used to visualize atherosclerotic plaques in Apoe−/− mice.

    (A) Left: Chemical structure of 7-123I-Nec-1 tracer. Right: Representative images of aortic en face (no stain and ORO stain) and autoradiography from mice injected with 123I-labeled Nec-1 tracer. (B) Mice were injected with nonradioactive Cl–Nec-1 compound 1.5 hours before being injected with radiolabeled 123I-Nec-1 tracer and subjected to autoradiography (images shown) and lesional uptake quantification (n = 3 mice per group). (C) Correlation of lesional uptake of ORO compared to 123I-Nec-1 (n = 9).

  • Fig. 5 Nec-1 therapy decreases atherosclerotic lesion progression and markers of instability in Apoe−/− mice.

    (A) Apoe−/− mice were fed a Western diet for 6 weeks before implantation of time-release pellets containing placebo or Nec-1s (2 mg/kg per day). After four additional weeks of Western diet feeding, the mice were harvested for morphometric analysis of atherosclerosis. (B) En face lesion area was measured in placebo- and Nec-1s–treated mice and is represented as lesion area as a percentage of total aorta area. (C) Lesion area in the aortic sinus in placebo- and Nec-1s–treated mice is represented as total area in μm2. (D) Necrotic core area within aortic sinus lesions. (E to G) Immunohistochemical staining of smooth muscle-α actin (SMC marker) (E), CD68 (macrophage marker) (F), and pMLKL (G) was performed on aortic sinus lesions and quantified with ImageJ. Representative images per group are shown. AU, arbitrary units. (H) Serum IL-1β in mice from placebo- or Nec-1–treated groups was measured at sacrifice by enzyme-linked immunosorbent assay (ELISA). *P ≤ 0.05, **P < 0.01 by Student’s t test.

  • Table 1 Body weight and serum cholesterol measurements in ApoE−/− mice.

    TC, total cholesterol; HDL, high-density lipoprotein.

    PlaceboNec-1P ≤ 0.05?
    Body weight (g)26.9 ± 4.930.4 ± 6.2ns
    TC (mg/dl)1109 ± 2191046 ± 166ns
    LDL (mg/dl)695 ± 173814 ± 183ns
    HDL (mg/dl)27 ± 1126 ± 15ns

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/2/7/e1600224/DC1

    fig. S1. Percentage of macrophage cell death with oxLDL treatment and Nec-1.

    fig. S2. Cell death in SMCs and macrophages treated with DAMPs.

    fig. S3. Analysis of ROS production and PGAM expression in macrophages.

  • Supplementary Materials

    This PDF file includes:

    • fig. S1. Percentage of macrophage cell death with oxLDL treatment and Nec-1.
    • fig. S2. Cell death in SMCs and macrophages treated with DAMPs.
    • fig. S3. Analysis of ROS production and PGAM expression in macrophages.

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