Reduced apoptosis and plaque necrosis in advanced atherosclerotic lesions of Apoe-/- and Ldlr-/- mice lacking CHOP - PubMed (original) (raw)

Reduced apoptosis and plaque necrosis in advanced atherosclerotic lesions of Apoe-/- and Ldlr-/- mice lacking CHOP

Edward Thorp et al. Cell Metab. 2009 May.

Abstract

Endoplasmic reticulum (ER) stress is a hallmark of advanced atherosclerosis, but its causative role in plaque progression is unknown. In vitro studies have implicated the ER stress effector CHOP in macrophage apoptosis, a process involved in plaque necrosis in advanced atheromata. To test the effect of CHOP deficiency in vivo, aortic root lesions of fat-fed Chop+/+;Apoe-/- and Chop-/-;Apoe-/- mice were analyzed for size and morphology. Despite similar plasma lipoproteins, lesion area was 35% smaller in Chop-/-;Apoe-/- mice. Most importantly, plaque necrosis was reduced by approximately 50% and lesional apoptosis by 35% in the CHOP-deficient mice. Similar results were found in fat-fed Chop-/-;Ldlr-/- versus Chop+/+;Ldlr-/- mice. Thus, CHOP promotes plaque growth, apoptosis, and plaque necrosis in fat-fed Apoe-/- and Ldlr-/- mice. These data provide direct evidence for a causal link between the ER stress effector CHOP and plaque necrosis and suggest that interventions weakening this arm of the UPR may lessen plaque progression.

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Figures

Figure 1

Figure 1. Total Body Weight, Plasma Lipoproteins, and CHOP Expression in Chop+/+;_Apoe_−/− and _Chop_−/−;_Apoe_−/− Mice

(A–B) Body weight and total plasma cholesterol of Western diet-fed male Chop+/+;_Apoe_−/− and _Chop_−/−;_Apoe_−/− mice (n = 20 and 21, respectively). (C) Pooled plasma samples were subjected to fast performance liquid chromatography gel-filtration, and the fractions were assayed for cholesterol concentration. None of the differences between the two groups of mice in A–C were statistically significant. (D) Peritoneal macrophages from Chop+/+;_Apoe_−/− and _Chop_−/−;_Apoe_−/− mice were loaded with lipoprotein-derived unesterified cholesterol for the indicated times, and then whole-cell lysates were subjected to immunoblot analysis for CHOP and, as a loading control, tubulin. (E) RNA from the intima of aortic root lesions of Chop+/+;_Apoe_−/− and _Chop_−/−;_Apoe_−/− mice (see Figure 2) was captured by LCM, and Chop and Cypa mRNA were quantified by RT-QPCR. Relative expression was normalized to Cypa. Results are displayed as the mean ± S.E.M. (n = 3 RT-PCR replicates). **, Chop mRNA not detected.

Figure 2

Figure 2. Reduction in Aortic Root Lesion Area and Necrotic Area in _Chop_−/−;_Apoe_−/− Mice

(A) The images show representative sections of aortic roots from each group of mice were stained with hematoxylin and eosin. Quantification was conducted on lesions from 20 Chop+/+;_Apoe_−/− mice and 21 _Chop_−/−;_Apoe_−/− mice. *, P = 0.03. (B) Representative sections of hematoxylin and eosin-stained aortic root sections from Chop+/+;_Apoe_−/− and _Chop_−/−;_Apoe_−/− mice. (*= necrotic areas). The bar graph shows quantification of anuclear, afibrotic, and eosin-negative necrotic areas (n = 20 for both groups of mice). **, P < 0.01. (C) Quantification of necrotic area in a subgroup of 12 Chop+/+;_Apoe_−/− lesions and 10 _Chop_−/−;_Apoe_−/− lesions with statistically identical lesion area. *, P = 0.039.

Figure 3

Figure 3. Reduction in Peritoneal Macrophage and Aortic Root Lesional Macrophage Apoptosis in _Apoe_−/−;_Chop_−/− Mice

(A) Left graph, Macrophages from Chop+/+;_Apoe_−/− or _Chop_−/−;_Apoe_−/− mice were loaded with lipoprotein-derived unesterified cholesterol and then assayed for apoptosis. *, P < 0.01. _Middle graph_, Macrophages were incubated for 18 h with vehicle control (0.2% ethanol) or 50 μM 7-ketocholesterol and then assayed for apoptosis. *, _P_<0.01 compared with _Chop_+/+;_Apoe_−/− macrophages. _Right graph_, Macrophages were incubated for 15 h with vehicle control, 50 μg/ml oxidized LDL (_OxLDL_), 1 mM SIN-1, or both compounds. The cells were then assayed for apoptosis. _Asterisk_ = _P_<0.01 compared with _Chop_+/+;_Apoe_−/− macrophages. In the case of cholesterol loading, ~5% of apoptotic cells were PI-positive. With 7-ketocholesterol and OxLDL + SIN-1, the percentage of PI-positive cells was >50%, indicating advanced apoptosis, and both annexin-positive and PI-positive cells were decreased in the CHOP–deficient macrophages. (B) Efferocytosis assays were conducted using various combinations of peritoneal macrophages from Chop+/+;_Apoe_−/− and _Chop_−/−;_Apoe_−/− mice, where the macrophages served as the source of either the apoptotic cells or the efferocytes. None of the differences shown are statistically significant. (C) Representative micrographs show less TUNEL-positive signal (red; arrows) in nuclei (blue) of aortic root lesions from _Chop_−/−;_Apoe_−/− lesions. Bar, 10 μm. Quantification of TUNEL-positive nuclei was conducted on lesions from 20 Chop+/+;_Apoe_−/− mice and 21 _Chop_−/−;_Apoe_−/− mice. *, P<0.01. (D) Representative micrographs show less activated caspase-3 (red; arrows) in aortic root lesions from _Chop_−/−;_Apoe_−/− _vs. Chop_−/−;_Apoe_−/− lesions. Bar, 10 μm. Quantification of TUNEL-positive cells was conducted on lesions from 7 Chop+/+;_Apoe_−/− mice and 7 _Chop_−/−;_Apoe_−/− mice. *, P<0.05. (E) Representative sequential sections of a Chop+/+;_Apoe_−/− lesion stained for nuclei (Hoechst), TUNEL, and macrophages. Bar, 10 μm.

Figure 4

Figure 4. Reduction in Aortic Root Lesion Area, Necrotic Area, and Apoptosis in _Ldlr_−/−;_Chop_−/− Mice

(A) Body weight, plasma total cholesterol, plasma HDL cholesterol, and FPLC plasma cholesterol profile of 12-wk Western diet-fed male Chop+/+;_Ldlr_−/− and _Chop_−/−;_Ldlr_−/− mice (n = 12 and 17, respectively). None of the differences between the two groups of mice in these parameters were statistically significant. (B), Quantification of total aortic root lesion area and necrotic area of the mice described in A. *, P<0.05. (C) Representative micrographs and quantification of TUNEL-positive signal (red) in nuclei (blue) of aortic root lesions from _Chop_−/−;_Ldlr_−/− lesions. Bar, 10 μm. *, P<0.05. (D) Quantification of activated caspase-3 in aortic root lesions from Chop+/+;_Ldlr_−/− and _Chop_−/−;_Ldlr_−/ lesions. *, P<0.05.

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