Periadventitial Inducible Nitric Oxide Synthase Expression and Intimal Thickening

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Recent evidence indicates that the protein synthesis inhibitor cycloheximide triggers selective macrophage death in rabbit atheroma-like lesions without affecting smooth muscle cells (SMCs) or the endothelium, thereby favoring a stable plaque phenotype. In this study, we report that puromycin, a protein synthesis inhibitor with a different mode of action but with similar ability to inhibit de novo protein synthesis, did not reveal plaque-stabilizing effects. The macrophage and the SMC content readily decreased in puromycin-treated atheroma-like lesions in rabbit carotid arteries. Moreover, puromycin induced apoptosis in macrophages and SMCs in vitro. Puromycintreated SMCs showed signs of endoplasmic reticulum (ER) stress, as demonstrated by CCAAT/enhancer-binding protein homologous protein (CHOP) protein expression, splicing of X-box-binding protein 1 mRNA, and phosphorylation of eukaryotic translation initiation factor 2␣. The ER stress inducer thapsigargin up-regulated CHOP protein expression in SMCs without affecting their viability, indicating that ER stress not necessarily results in cell death. Puromycin, but not thapsigargin, activated the ER stress-related caspase-12. Treatment of SMCs with a combination of cycloheximide and puromycin inhibited ER stress and partially improved SMC viability. In addition, puromycin, but not cycloheximide or thapsigargin, induced intracellular accumulation of polyubiquitinated proteins in SMCs, whereas the proteasome function was not affected. Taken together, puromycin, in contrast to cycloheximide, induces SMC apoptosis, thereby favoring an unstable plaque phenotype. SMC death upon puromycin treatment could only be partially prevented by cycloheximide, which completely blocked ER stress. However, other or additional mechanisms, such as increased polyubiquitination of proteins, might be involved in puromycin-induced SMC death.

Section: Methodsmentioning

confidence: 99%

Differential Effect of the Protein Synthesis Inhibitors Puromycin and Cycloheximide on Vascular Smooth Muscle Cell Viability

Croons

Martinet²,

Herman³

et al. 2008

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“…Formalin-fixed carotid artery rings were paraffin-embedded and stained with hematoxylin/eosin or Verhoef 's elastin. Immunohistochemical detection was carried out using an indirect antibody conjugate technique (Kockx et al, 1992;De Meyer et al, 2000). The following primary antibodies were used: anti-␣-SMC actin (clone 1A4; Sigma-Aldrich) and MO/RAM11 (antirabbit macrophages; Dako Denmark A/S) on paraffin sections; anti-CD31 (JC/70A; Dako Denmark A/S) and anti-FITC (polyclonal antifluorescein isothiocyanate horse radish peroxidase conjugated; Dako Denmark A/S) on frozen sections.…”

Section: Methodsmentioning

confidence: 99%

Selective Clearance of Macrophages in Atherosclerotic Plaques by the Protein Synthesis Inhibitor Cycloheximide

Croons¹,

Martinet²,

Herman³

et al. 2006

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Macrophages are an essential component of unstable atherosclerotic plaques and play a pivotal role in the destabilization process. We have demonstrated previously that local delivery of the mammalian target of rapamycin (mTOR) inhibitor everolimus selectively clears macrophages in rabbit plaques. Because mTOR controls mRNA translation, inhibition of protein synthesis might induce selective macrophage cell death. We therefore investigated in the present study the effect of the protein synthesis inhibitor cycloheximide on macrophage and smooth muscle cell (SMC) viability. In vitro studies with cultured macrophages and SMCs showed that cycloheximide induced selective apoptosis of macrophages in a concentration-and time-dependent manner. Moreover, macrophages could be selectively depleted in rabbit carotid artery rings with collarinduced atherosclerotic plaques after in vitro treatment with cycloheximide. Local in vivo administration of cycloheximide via osmotic minipumps to rabbit carotid arteries with collarinduced atherosclerotic plaques significantly reduced the macrophage but not the SMC content. Cycloheximide-treated plaques showed signs of apoptosis (increased terminal deoxynucleotidyl transferase end labeling and fluorescein isothiocyanate-Val-Ala-DL-Asp(O-methyl)-fluoromethylketone labeling) that did not colocalize with SMCs. Organ chamber studies demonstrated that the functionality of SMCs and the endothelium were not influenced by cycloheximide treatment. All together, these findings demonstrate that cycloheximide decreases the macrophage load in atherosclerotic plaques by induction of apoptosis without changing SMC content or contractility.

“…Formalin-fixed carotid artery rings were paraffin-embedded and stained with hematoxylin/eosin (H/E) or Verhoeff's elastin. Immunohistochemical detection was carried out using an indirect antibody conjugate technique (Kockx et al, 1992;De Meyer et al, 2000). The following mouse primary antibodies were used: anti-␣-SMC actin (clone 1A4; Sigma-Aldrich) and anti-rabbit macrophage (clone RAM11; DakoCytomation Denmark A/S) on paraffin sections and anti-CD31 (JC/70A; DakoCytomation Denmark A/S) on frozen sections.…”

Section: Role Of Mapk In Anisomycin-induced Macrophage Apoptosismentioning

confidence: 99%

The Protein Synthesis Inhibitor Anisomycin Induces Macrophage Apoptosis in Rabbit Atherosclerotic Plaques through p38 Mitogen-Activated Protein Kinase

Croons

Martinet²,

Herman³

et al. 2009

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Because macrophages play a major role in atherosclerotic plaque destabilization, selective removal of macrophages represents a promising approach to stabilize plaques. We showed recently that the protein synthesis inhibitor cycloheximide, in contrast to puromycin, selectively depleted macrophages in rabbit atherosclerotic plaques without affecting smooth muscle cells (SMCs). The mechanism of action of these two translation inhibitors is dissimilar and could account for the differential effects on SMC viability. It is not known whether selective depletion of macrophages is confined to cycloheximide or whether it can also be achieved with translation inhibitors that have a similar mechanism of action. Therefore, in the present study, we investigated the effect of anisomycin, a translation inhibitor with a mechanism of action similar to cycloheximide, on macrophage and SMC viability. In vitro, anisomycin induced apoptosis of macrophages in a concentration-dependent manner, whereas SMCs were only affected at higher concentrations. In vivo, anisomycin selectively decreased the macrophage content of rabbit atherosclerotic plaques through apoptosis. The p38 mitogen-activated protein kinase (MAPK) inhibitor SB202190 [4-(4-fluorophenyl)-2-(4-hydroxyphenyl)-5-(4-pyridyl)-1H-imidazole] prevented anisomycin-induced macrophage death, without affecting SMC viability. SB202190 decreased anisomycin-induced p38 MAPK phosphorylation, did not alter c-Jun NH 2 -terminal kinase (JNK) phosphorylation, and increased extracellular signal-regulated kinase (ERK) 1/2 phosphorylation. The latter effect was abolished by the mitogenactivated protein kinase kinase 1/2 inhibitor U0126 [1,4-diamino-2,3-dicyano-1,4-bis(2-aminophynyltio)butadiene ethanolate], although the prevention of anisomycin-induced macrophage death by SB202190 remained unchanged. The JNK phosphorylation inhibitor SP600125 did not affect anisomycin-induced macrophage or SMC death. In conclusion, anisomycin selectively decreased the macrophage content in rabbit atherosclerotic plaques, indicating that this effect is not confined to cycloheximide. p38 MAPK, but not ERK1/2 or JNK, plays a major role in anisomycin-induced macrophage death.