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. 2009 Dec;15(12):1383-91.
doi: 10.1038/nm.2067. Epub 2009 Nov 29.

Reducing endoplasmic reticulum stress through a macrophage lipid chaperone alleviates atherosclerosis

Ebru Erbay  1 , Vladimir R Babaev, Jared R Mayers, Liza Makowski, Khanichi N Charles, Melinda E Snitow, Sergio Fazio, Michelle M Wiest, Steven M Watkins, Macrae F Linton, Gökhan S Hotamisligil
Affiliations

Reducing endoplasmic reticulum stress through a macrophage lipid chaperone alleviates atherosclerosis

Ebru Erbay et al. Nat Med. 2009 Dec.

Erratum in

  • Nat Med. 2010 Feb;16(2):237

Abstract

Macrophages show endoplasmic reticulum (ER) stress when exposed to lipotoxic signals associated with atherosclerosis, although the pathophysiological importance and the underlying mechanisms of this phenomenon remain unknown. Here we show that mitigation of ER stress with a chemical chaperone results in marked protection against lipotoxic death in macrophages and prevents macrophage fatty acid-binding protein-4 (aP2) expression. Using genetic and chemical models, we show that aP2 is the predominant regulator of lipid-induced macrophage ER stress. The absence of lipid chaperones incites an increase in the production of phospholipids rich in monounsaturated fatty acids and bioactive lipids that render macrophages resistant to lipid-induced ER stress. Furthermore, the impact of aP2 on macrophage lipid metabolism and the ER stress response is mediated by upregulation of key lipogenic enzymes by the liver X receptor. Our results demonstrate the central role for lipid chaperones in regulating ER homeostasis in macrophages in atherosclerosis and show that ER responses can be modified, genetically or chemically, to protect the organism against the deleterious effects of hyperlipidemia.

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Figures

Figure 1
Figure 1. PBA treatment protects against macrophage ER stress and reduces vascular disease progression
(a–d) We induced ER stress in WT macrophages by 500 µM PA or 300 nM thapsigargin (Thaps) in the presence or absence of 3 mM PBA. P-PERK and P-eIF2–α were examined by Western blotting (a), mRNA levels of Ddit3 (b) and sXBP-1 were examined by qRT-PCR (c), and apoptosis were examined by TUNEL assays (d) (data represent mean±SEM; * p<0.05). (e–g) Atherosclerotic lesion area in the aortic sinus from mice treated with either control or PBA for 2 weeks (n≥13) is reported (data represents mean±SD; * indicates p<0.05) (e) and the serial sections were stained with antibodies against P-PERK (f), P-eIF2–α (f), ATF3 (g) and MOMA-2 (f,g) (Red indicates positive staining with antibody. Scale bars represent 200 µm). (g) Relative fluorescent intensity was calculated for antibody staining corresponding to ATF3 and P- eIF2–α expression (n≥3) in the macrophage-dense areas (data represent mean±SD; * indicates p<0.05). (h) Percent of apoptotic cells (TUNEL positive) in the aortic sinus area are shown for PBS (control) or 100 mg kg−1 PBA (PBA-100) treated mice (Arrows point to apoptotic cells. Scale bars represent 200 µm. *indicates p<0.05).
Figure 2
Figure 2. Requirement for aP2 in lipid-induced ER stress and apoptosis
(a–b) Serial sections from aortic sinus of PBS or PBA treated ApoE−/− mice were stained for antibodies against MOMA-2 or aP2 (Arrows point to red staining for these antibodies in macrophage-dense areas. Scale bar represents 200 µm) (b) Relative fluorescence intensity for aP2 expression (green) in the macrophage-dense areas (red) of the lesions was calculated (Scale bar represents 100 µm. Data represent mean±SD; * indicates p<0.05 and n≥3). (c) aP2 expression was determined by Western blotting in PA (500 µM) treated macrophages in the presence or absence of 3 µM PBA or 100 µg mL−1 cycloheximide (CHX). (d) P-PERK, P-eIF2–α was examined by Western blotting and JNK activity (P-cJun) by an in vitro kinase assay from PA (500 µM) treated macorphages. (e) sXBP-1 was examined by qRT-PCR in (500 µM) PA- or (2 µg/mL) tunicamycin (Tunic) treated (12 h) macrophages. (f) Cleaved PARP and tubulin expression were examined by Western blotting in PA-treated (24 h) macrophages (relative band intensities were quantified and data represent mean±SEM; *p<0.05). (g) P-PERK, P-eIF2–α were examined by Western blotting, JNK activity (P-c-Jun) by a kinase assay, and sXBP-1 by RT-PCR from macrophages treated with 10 µM ACAT inhibitor (Ai) and 100 µg/mL Ac-LDL (24 hours) or 300 nM thapsgiargin (Thaps) (The ratio of relative intensities corresponding to spliced (s-XBP1) and unspliced (u-XBP1) were calculated).
Figure 3
Figure 3. aP2 deficiency protects from hypercholesteremia induced macrophage ER stress and apoptosis in atherosclerotic lesions
(a–b) Immunohistochemical staining with antibodies against MOMA-2, P-PERK, P-eIF2–α (a,b), CHOP (a), and ATF3 (b) were performed in atherosclerotic lesions from the proximal aorta of ApoE−/− and aP2−/−ApoE−/− mice fed a Western diet for 16 weeks (Arrows point to ATF3 and P-eIF2–α (green), expressed in the MOMA-2 positive (red) areas of the lesions. Scale bars in (a) represent 50 µm and in (b) represent 100 µm). (c) Relative fluorescent intensity was calculated for stainings corresponding to ATF3 and P-eIF2–α in the macrophage-dense areas (data represent mean±SD; * indicates p<0.05, n≥3). (d–e) Apoptotic macrophages in the lesions from ApoE−/− and aP2−/−ApoE−/− mice were determined by TUNEL assay (Arrows point to apoptotic cells. Scale bars represent 100 µm. * indicates p<0.05). (f–g) Macrophage lines were stressed with PA (500 µM) in the presence of vehicle (−) or varying does of the aP2-i (0.1–50 µM). P-PERK and P-eIF2–α was examined by Western blotting (f) and sXBP-1 and Ddit3 mRNA were analyzed by qRT-PCR from macrophages treated with 25 µM of aP2-i (g). (h) Double immunofluorescent staining was performed using antibodies against MOMA-2 and ATF-3 in the atherosclerotic lesions from ApoE−/− mice treated with vehicle or aP2-i (15 mg kg−1 for 14 weeks) (Arrows indicate staining for ATF3 (green) in MOMA-2 positive areas (red). Scale bars represent 100µm).
Figure 4
Figure 4. Regulation of macrophage lipid composition by aP2
(a) Lipid class composition analysis for TG, PL and FA was performed. The F statistics from one-way ANOVA are displayed as red diamonds over the distribution of F statistics from permuted data. The black line is the 95th percentile of the F statistics over 1000 permutations. The higher the value of the F-statistics from ANOVA, the more different the groups are. The heat map displays the observed data, centered to the mean of the WT genotype and scaled by the standard deviation of all observations. (b) The mean concentration of C16:1n7 and C18:1n7 was determined for each lipid class in the various macrophage lines. (c–d) Total lipid composition: Percent total lipids (c) and bar plots of the mean concentration of lipids (d) in the macrophage lines.
Figure 5
Figure 5. A central role for SCD and C16:1n7 in aP2 mediated lipotoxic signaling
(A) A summary of the lipid changes that occur as a result of aP2 deficiency in macrophages (LCE and ELOVL; fatty acid elongase for long chain fatty acid). (b)SCD-1 mRNA levels were examined by qRT-PCR in primary peritoneal macrophages at the base line or (c) after treatment of animals aP2-i for 6 weeks (n=6) (data represent mean±SEM; * indicates p<0.05). (d–e) ER stress was induced in macrophages by PA (300 µM) or tunicamycin (2 mM) treatment for 3 hours. Cells were pretreated with PAO (300 µM) PAO, where indicated. P-PERK, P-eIF2–α and cleaved PARP were examined by Western blotting (d) and Ddit3 and sXBP-1 mRNA were examined by qRT-PCR (E). (F) From macrophage lines treated with SCD-1 siRNA (50–100 nM) or scrambled (−) siRNA, SCD activity (upper panel) was examined by an enzymatic assay and SCD protein expression (lower panel) was examined by Western blotting (G) P-PERK, P-eIF2–α and cleaved PARP were examined by Western blotting from lysates of aP2−/− macrophages treated with negative (−) siRNA or SCD-1 specific siRNA (100nM) and treated with or without PA (500 µM) (data represent mean±; SEM; * indicates p<0.05).
Figure 6
Figure 6. Linking toxic lipids to ER stress and atherosclerosis through aP2-LXR–α crosstalk
(a) Alignment of LXR responsive element (LXRE) on Fasn and SCD-1 promoters. (b) LXR–driven transcriptional activity was determined from various macrophage lines upon stimulation with a synthetic T0901317 (10 µM) or endogenous 25-hydroxycholesterol (10 µM) LXR ligand (luciferase activity is reported after normalizing to transfection efficiency). (c) Relative LXR–α and LXR–β protein levels in various macrophage lines were examined by Western blotting (d) Relative SCD-1 and Fasn mRNA levels from aP2−/− macrophage treated with a scrambled (−) or Nr1h3-specific siRNA were examined by qRT-PCR. (e) Lysates from various macrophages treated with scrambled (−) siRNA or a specific siRNA against SCD-1 or Nr1h3 and stressed with or without PA (500 µM) were examined for P-PERK and P-eIF2–α by Western blotting. (f) Lysates from peritoneal macrophages from aP2−/−Nr1h3−/− or WT mice stressed with or without PA (500 µM) were examined for P-PERK, P-eIF2–α and cleaved PARP by Western blotting. (g) A cellular lipotoxicity model: Toxic levels of lipids are sensed by the ER through an aP2-dependent pathway and induce the UPR and lead to macrophage apoptosis. The absence of aP2 serves to reactivate macrophage de novo lipogenesis pathways and promotes desaturation, particularly through LXRα-mediated activation of SCD-1, leading to increased production of bioactive lipids and resistance to ER stress. Our findings indicate that alleviation of macrophage ER stress, either through aP2 inhibition or enhancing ER function, is protective against atherosclerosis.
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References

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