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

Abstract

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.

Involvement of mTOR kinase in cytokine-dependent microglial activation and cell proliferation

“…mTOR inhibitors were able to bring back the stimulatory effect of TII to control levels that may be considered as another evidence of the inhibitory effects of these drugs on cytokine-dependent microglial activation. However, microglial viability was significant affected by mTOR inhibition under baseline conditions, which is consistent with the findings that mTOR inhibition can lead to autophagic cell death [46] or to apoptosis [47]. Moreover, RAD reduced in a significant and dose-dependent manner microglial cell proliferation, assessed as the rate of BrdU incorporation into newly synthesized DNA in dividing cells.…”

“…Thus the effects of mTOR inhibitors seem to be cell specific. Similar findings were obtained in peripheral models, where RAD has been found to selectively reduce macrophage infiltration of atherosclerotic plaques without affecting smooth muscle cell viability [46]. At variance with the above evidence, it has been recently shown that mTOR may be involved in cell growth and reactive transformation of spinal cord astrocytes, particularly in response to epidermal growth factor [49].…”

Overexpression of Smooth Muscle Myosin Heavy Chain Leads to Activation of the Unfolded Protein Response and Autophagic Turnover of Thick Filament-associated Proteins in Vascular Smooth Muscle Cells

“…Cycloheximide, a protein translation inhibitor that is well tolerated by SMCs and does not cause cell death (17,18), was administered to block protein elongation, and contractile proteins levels were followed over time. In NTG cells, the contractile proteins remain highly stable for up to 48 h of exposure to cycloheximide (Fig.…”

Everolimus-Induced mTOR Inhibition Selectively Depletes Macrophages in Atherosclerotic Plaques by Autophagy

“…3). 12 Similarly, treatment of plaques with the nitric oxide donor molsidomine triggers endoplasmic reticulum stress, inhibition of de novo protein synthesis through hyperphosphorylation of eIF2a and selective macrophage apoptosis (Martinet W, unpublished results). 13 Measurements of oxygen consumption as well as immunodetection of markers for DNA synthesis/repair indicate that plaque macrophages are metabolically highly active and thus more sensitive to protein synthesis inhibitors as compared to SMCs.…”