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PRECLINICAL STUDY

Selective Clearance of Macrophages in Atherosclerotic Plaques by Autophagy

Stefan Verheye, MD, PhD^_*,_*, Wim Martinet, PhD, Mark M. Kockx, MD, PhD^,, Michiel W.M. Knaapen, PhD, Koen Salu, MD, PhD, Jean-Pierre Timmermans, PhD^||, Jeffrey T. Ellis, PhD^¶, Deborah L. Kilpatrick, PhD^¶ and Guido R.Y. De Meyer, PharMD, PhD

^_* Antwerp Cardiovascular Institute Middelheim, Antwerp, Belgium
Division of Pharmacology, University of Antwerp, Antwerp, Belgium
Department of Pathology, Middelheim Hospital, Antwerp, Belgium
Histogenex, Edegem, Belgium
^|| Laboratory of Cell Biology and Histology, University of Antwerp, Antwerp, Belgium
^¶ Guidant Corporation, Santa Clara, California

Manuscript received August 21, 2006; revised manuscript received September 26, 2006, accepted September 28, 2006.

^_* Reprint requests and correspondence: Dr. Stefan Verheye, Antwerp Cardiovascular Institute, Middelheim Hospital, Lindendreef 1, 2020 Antwerp, Belgium. (Email: stefan.verheye{at}pandora.be).

OBJECTIVES: The purpose of this study was to investigate whether stent-based delivery of an inhibitor of mammalian target of rapamycin (mTOR) can selectively clear macrophages in rabbit atherosclerotic plaques.

BACKGROUND: Current pharmacologic approaches to stabilize atherosclerotic plaques have only partially reduced the incidence of acute coronary syndromes and sudden death. Macrophages play a pivotal role in plaque destabilization, whereas smooth muscle cells (SMC) promote plaque stability.

METHODS: Stents eluting the mTOR inhibitor everolimus were implanted in atherosclerotic arteries of cholesterol-fed rabbits. In addition, in vitro experiments using explanted atherosclerotic segments and cultured macrophages as well as SMC were performed.

RESULTS: Stents eluting everolimus led to a marked reduction in macrophage content without altering the amount of SMC compared with polymer control stents. In vitro studies showed that everolimus treatment induced inhibition of translation in both cultured macrophages and SMC. However, cell death occurred only in macrophages and was characterized by bulk degradation of long-lived proteins, processing of microtubule-associated protein light chain 3, and cytoplasmic vacuolization, which are all markers of autophagy. Everolimus-induced autophagy was mediated by mTOR inhibition, because cell viability was not affected using tacrolimus, an mTOR-independent everolimus analog. Moreover, mTOR gene silencing was associated with selective induction of macrophage cell death. Autophagic macrophage cell death was confirmed by transmission electron microscopy both in cultured cells and in atherosclerotic explants.

CONCLUSIONS: Stent-based delivery of everolimus selectively cleared macrophages in rabbit atherosclerotic plaques by autophagy, an mTOR inhibition-dependent and novel mechanism to induce cell death in mammalian cells.

Abbreviations and Acronyms   4E-BP1 = 4E-binding protein 1   eEF2 = eukaryotic elongation factor 2   eIF2 = eukaryotic initiation factor 2 alpha   FKBP12 = FK506-binding protein 12   LC-3 = microtubule-associated protein light chain 3   mTOR = mammalian target of rapamycin   SMC = smooth muscle cells

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