Nitric oxide selectively depletes macrophages in atherosclerotic plaques via induction of endoplasmic reticulum stress

Abstract

Background and purpose: Macrophages in atherosclerotic plaques have a tremendous impact on atherogenesis and plaque destabilization. We previously demonstrated that treatment of plaques in cholesterol-fed rabbits with the nitric oxide (NO) donor molsidomine preferentially eliminates macrophages, thereby favouring features of plaque stability. In this study, we investigated the underlying mechanism.

Experimental approach: Macrophages and smooth muscle cells (SMCs) were treated in vitro with the NO donors, spermine NONOate or S-nitroso-N-acetylpenicillamine (SNAP) as well as with the well-known endoplasmic reticulum (ER) stress inducers thapsigargin, tunicamycin, dithiothreitol or brefeldin A. Cell viability was analysed by Neutral Red viability assays. Cleavage of caspase-3, DNA fragmentation and ultrastructural changes were examined to characterize the type of macrophage death. Induction of ER stress was evaluated by measuring C/EBP homologous protein (CHOP) expression, phosphorylation of eukaryotic initiation factor 2 alpha (eIF2a), splicing of X-box binding protein 1 (XBP1) and inhibition of protein synthesis.

Key results: Macrophages and SMCs treated with spermine NONOate or SNAP showed several signs of ER stress, including upregulation of CHOP expression, hyperphosphorylation of eIF2 alpha, inhibition of de novo protein synthesis and splicing of XBP1 mRNA. These effects were similar in macrophages and SMCs, yet only macrophages underwent apoptosis. Plaques from molsidomine-treated atherosclerotic rabbits showed a 2.7-fold increase in CHOP expression as compared to placebo. Beside NO, selective induction of macrophage death could be initiated with thapsigargin and tunicamycin.

Conclusions and implications: Induction of ER stress explains selective depletion of macrophages in atherosclerotic plaques by a NO donor, probably via inhibition of protein synthesis.

Figure 1

Effect of the nitric oxide donors spermine NONOate and S-nitroso-N-acetylpenicillamine (SNAP) on viability of J774A.1 macrophages (MΦ), thioglycolate elicited peritoneal macrophages and smooth muscle cells (SMCs). Cells were exposed to various concentrations of spermine NONOate (a) or SNAP (b) in serum-containing medium for 24 h. Cell death was examined by Neutral Red viability assays. Results represent the mean±s.e.mean of three independent experiments. ^**P<0.01 versus untreated cells (ANOVA, followed by Dunnett's test).

Figure 2

Characterization of spermine NONOate-induced cell death in J774A.1 macrophages. Cells were treated with 300 μM spermine NONOate for up to 24 h. Cleavage of procaspase-3 (procasp-3) and internucleosomal DNA fragmentation was analysed using western blotting (top) and agarose gel electrophoresis (bottom), respectively (a). Ultrastructural features of J774A.1 cells before (b) and after treatment with 300 μM spermine NONOate for 3 h (c) were analysed by electron microscopy. Spermine NONOate-treated cells were characterized by chromatin condensation (arrow) and blebbing of the plasma membrane (arrowheads). N indicates nucleus. Scale bar=2 μm. Results are representative images of three independent experiments.

Figure 3

Induction of endoplasmic reticulum (ER) stress in J774A.1 macrophages (MΦ) and smooth muscle cells (SMCs) treated with 300 μM spermine NONOate for up to 24 h. Expression of the ER stress protein C/EBP homologous protein (CHOP) as well as phosphorylation of eukaryotic initiation factor 2α (P-elF2α) was analysed by western blotting (a). Relative expression of CHOP mRNA (b) and splicing of X-box-binding protein 1 (XBP1) mRNA (c) was examined via real-time quantitative reverse transcription (RT)–PCR or conventional RT–PCR, respectively. ^*P<0.05; ^**P<0.01 versus untreated cells (ANOVA, followed by Dunnett's test). Results are representative of three independent experiments.

Figure 4

Inhibition of de novo protein synthesis in J774A.1 macrophages (MΦ) and smooth muscle cells (SMCs) after treatment with spermine NONOate (NO) or thapsigargin. Cells were treated with 300 μM spermine NONOate or 1 μM thapsigargin for 1 h in serum-containing medium and then pulse-labelled for 1 h with L-³⁵S-methionine/cysteine. Labelled proteins were measured by scintillation counting. Results represent the mean±s.e.mean of three independent experiments. ^**P<0.01 versus control (ANOVA, followed by Dunnett's test).

Figure 5

Spermine NONOate-induced cell death in J774A.1 macrophages after phagocytosis of aggregated low-density lipoprotein (agLDL). (a and b) Oil Red O staining of J774A.1 cells grown in the absence (a) or presence (b) of 200 μg/ml agLDL for 20 h. Scale bar=10 μm. (c) Viability of agLDL-laden and control J774A.1 cells after exposure to different concentrations of spermine NONOate. Results represent the mean±s.e.mean of three independent experiments. ^*P<0.05 versus cells without agLDL (unpaired Student's t-test).

Figure 6

Selective induction of J774A.1 macrophage cell (MΦ) death by the endoplasmic reticulum (ER) stress inducer thapsigargin. (a) Viability of MΦ and smooth muscle cells (SMCs) after exposure to different concentrations of thapsigargin for 24 h. ^**P<0.01 versus untreated cells (ANOVA, followed by Dunnett's test). (b–e) Cells were treated with 1 μm thapsigargin for the time indicated. Thapsigargin-induced J774A.1 cell death was characterized by cleavage of procaspase-3 (procasp-3) and internucleosomal DNA fragmentation (b). Western blot of eukaryotic initiation factor 2α (eIF2α) phosphorylation and C/EBP homologous protein (CHOP) expression (c), real-time reverse transcription (RT)–PCR analysis of CHOP mRNA (d), ^*P<0.05; ^**P<0.01 versus untreated cells (one-sample t-test), as well as splicing of X-box-binding protein 1 (XBP1) mRNA (e) was performed to document induction of ER stress. Results are representative of three independent experiments.

Figure 7

Viability of J774A.1 macrophages (MΦ) and smooth muscle cells (SMCs) after exposure to different concentrations of the endoplasmic reticulum stress inducers tunicamycin, dithiothreitol and brefeldin A for 24 h in serum-containing medium. Results represent the mean±s.e.mean of three independent experiments. ^*P<0.05; ^**P<0.01 versus untreated cells (ANOVA, followed by Dunnett's test).