Cryotherapy increases features of plaque stability in atherosclerotic rabbits.

AIMS: In the last 10 years, cryotherapy has been investigated as a new technology to treat vascular disease. The efficiency of cryotherapy in stabilising atherosclerotic plaques has never been described. The purpose of the present study was to evaluate the effect of catheter-based cryotherapy on atherosclerotic plaque composition in a rabbit model of atherosclerosis. METHODS AND RESULTS: Twenty-four New Zealand white rabbits were fed a 0.3% cholesterol-supplemented diet for 24 weeks. At two predefined sites of the atherosclerotic thoracic aorta, catheter-based cryotherapy, applying either single-dose, double-dose cryotherapy or control inflation, was performed after randomisation. Rabbits were continued on a cholesterol-supplemented diet for one day (acute) or four weeks (chronic). One day after cryotherapy, apoptotic cell death of smooth muscle cells (SMCs) and endothelial cells (ECs) was observed, whereas macrophages were unaffected. Four weeks later, the amount of SMCs was restored, the EC layer was regenerated, and a subendothelial macrophage-free layer was formed, indicative of a more stable plaque. In addition, both the thickness and the type I collagen content of the fibrous cap were increased. CONCLUSIONS: The present study demonstrated that cryotherapy is feasible and appears to stabilise atherosclerotic plaques in a rabbit model.

Drug-induced macrophage autophagy in atherosclerosis: for better or worse?

Autophagy is a reparative, life-sustaining process by which cytoplasmic components are sequestered in double membrane vesicles and degraded upon fusion with lysosomal compartments. Mice with a macrophage-specific deletion of the essential autophagy gene Atg5 develop plaques with increased apoptosis and oxidative stress as well as enhanced plaque necrosis. This finding indicates that basal autophagy in macrophages is anti-apoptotic and present in atherosclerotic plaques to protect macrophages against various atherogenic stressors. However, autophagy is impaired in advanced stages of atherosclerosis and its deficiency promotes atherosclerosis in part through activation of the inflammasome. Because basal autophagy can be intensified selectively in macrophages by specific drugs such as mammalian target of rapamycin (mTOR) inhibitors or Toll-like receptor 7 (TLR7) ligands, these drugs were recently tested as potential plaque stabilizing compounds. Stent-based delivery of the mTOR inhibitor everolimus promotes a stable plaque phenotype, whereas local administration of the TLR7 ligand imiquimod stimulates inflammation and plaque progression. Therefore, more drugs capable of inducing autophagy should be tested in plaque macrophages to evaluate the feasibility of this approach. Given that drug-induced macrophage autophagy is associated with pro-inflammatory responses due to cytokine release, induction of postautophagic necrosis or activation of phagocytes after clearance of the autophagic corpse, cotreatment with anti-inflammatory compounds may be required. Overall, this review highlights the pros and cons of macrophage autophagy as a drug target for plaque stabilization.

Toll-like receptor 7 stimulation by imiquimod induces macrophage autophagy and inflammation in atherosclerotic plaques.

Atherosclerotic plaques tend to rupture as a consequence of a weakened fibrous cap, particularly in the shoulder regions where most macrophages reside. Macrophages express Toll-like receptors to recognize pathogens and eliminate intracellular pathogens by inducing autophagy. Because Toll-like receptor 7 (TLR7) is thought to be expressed in macrophages but not in smooth muscle cells (SMCs), we investigated whether induction of macrophage autophagic death by TLR7 ligand imiquimod can affect the composition of atherosclerotic plaques in favor of their stability. Immunohistochemical staining of human carotid plaques as well as Western blotting of cultured macrophages and SMCs confirmed that TLR7 was expressed in macrophages, but not in SMCs. In vitro experiments showed that only TLR7 expressing cells underwent imiquimod-induced cell death, which was characterized by autophagosome formation. Imiquimod-treated macrophages activated nuclear factor-κB (NF-κB) and released pro-inflammatory cytokines and chemokines. This effect was inhibited by the glucocorticoid dexamethasone. Imiquimod-induced cytokine release was significantly decreased in autophagy-deficient macrophages because these cells died by necrosis at an accelerated pace. Local in vivo administration of imiquimod to established atherosclerotic lesions in rabbit carotid arteries induced macrophage autophagy without induction of cell death, and triggered cytokine production, upregulation of vascular adhesion molecule-1, infiltration of T-lymphocytes, accumulation of macrophages and enlargement of plaque area. Treatment with dexamethasone suppressed these pro-inflammatory effects in vivo. SMCs and endothelial cells in imiquimod-treated plaques were not affected. In conclusion, imiquimod induces macrophage autophagy in atherosclerotic plaques, but stimulates plaque progression through cytokine release and enhanced infiltration of inflammatory cells.

Everolimus triggers cytokine release by macrophages: rationale for stents eluting everolimus and a glucocorticoid.

OBJECTIVE: Stent-based delivery of the mammalian target of rapamycin (mTOR) inhibitor everolimus is a promising strategy for the treatment of coronary artery disease. We studied potential adverse effects associated with mTOR inhibition. METHODS AND RESULTS: Macrophages in culture were either treated with everolimus or starved to inhibit mTOR. Everolimus led to inhibition of protein translation, activation of p38 MAPK, and the release of proinflammatory cytokines (eg, IL-6, TNFα) and chemokines (eg, MCP1, Rantes) before induction of autophagic death. These effects were also observed with rapamycin, but not after starvation. Everolimus-induced cytokine release was similar in macrophages lacking the essential autophagy gene Atg7 but was inhibited when macrophages were cotreated with p38 MAPK inhibitor SB202190 or the glucocorticoid clobetasol. Combined stent-based delivery of clobetasol and everolimus in rabbit plaques downregulated TNFα expression as compared with everolimus-treated plaques but did not affect the ability of everolimus to induce macrophage clearance. CONCLUSIONS: mTOR inhibition by everolimus triggers cytokine release in macrophages through inhibition of protein translation and p38 activation. These findings provide a rationale for combined local treatment of atherosclerotic plaques with everolimus and an anti-inflammatory agent.

Therapeutic strategies to deplete macrophages in atherosclerotic plaques.

Macrophages can be found in all stages of atherosclerosis and are major contributors of atherosclerotic plaque development, progression and destabilization. Continuous recruitment of monocytes drives this chronic inflammatory disease, which can be intervened by several strategies: reducing the inflammatory stimulus by lowering circulating lipids and promoting cholesterol efflux from plaque, direct and indirect targeting of adhesion molecules and chemokines involved in monocyte adhesion and transmigration and inducing macrophage death in atherosclerotic plaques in combination with anti-inflammatory drugs. This review discusses the outlined strategies to deplete macrophages from atherosclerotic plaques to promote plaque stabilization.

Inhibition of inositol monophosphatase by lithium chloride induces selective macrophage apoptosis in atherosclerotic plaques.

BACKGROUND AND PURPOSE: Lithium chloride (LiCl) inhibits inositol monophosphatase (IMPase) at therapeutic concentrations. Given that LiCl induces death in cultured macrophages and that macrophages play an active role in atherosclerotic plaque destabilization, we investigated whether LiCl would induce selective macrophage death to stabilize the structure of the plaque. EXPERIMENTAL APPROACH: The effect of LiCl was assessed on macrophages and smooth muscle cells (SMCs) in culture, in isolated atherosclerotic carotid arteries from rabbits and after local in vivo treatment via osmotic minipumps to rabbits with collared atherosclerotic carotid arteries. In addition, in vitro experiments were performed to elucidate the mechanism of LiCl-induced macrophage death. KEY RESULTS: In vitro, whereas SMCs were highly resistant, LiCl induced macrophage death characterized by externalization of phosphatidylserine, caspase-3 cleavage and DNA fragmentation, all indicative of apoptosis. LiCl reduced inositol-1,4,5-trisphosphate levels in macrophages. Moreover, the IMPase inhibitor L-690 330 as well as IMPase gene silencing induced macrophage apoptosis. Both in vitro treatment of rabbit atherosclerotic carotid arteries with LiCl and local in vivo administration of LiCl to the plaques decreased plaque macrophages through apoptosis, as shown by terminal deoxynucleotidyl transferase deoxyuridine triphosphate (dUTP) nick-end labelling (TUNEL), without affecting SMCs. Vasomotor studies in vitro showed that LiCl did not affect the functionality of SMCs and endothelial cells. CONCLUSIONS AND IMPLICATIONS: LiCl selectively decreased the macrophage load in rabbit atherosclerotic plaques via IMPase inhibition without affecting the viability or functionality of SMCs and endothelial cells. These data provide evidence for local administration of an IMPase inhibitor to stabilize atherosclerotic plaques.

Cell death-mediated cleavage of the attraction signal p43 in human atherosclerosis: implications for plaque destabilization.

OBJECTIVE: Apoptosis is a key feature of advanced atherosclerotic plaques. Attraction signals such as p43 released from apoptotic cells play a crucial role in the timely removal of the apoptotic remnants by recruiting fresh phagocytes. Here, we sought to determine whether p43 may link apoptosis to inflammation and plaque progression. METHODS AND RESULTS: RT-PCR and immunohistochemistry showed that p43 was abundantly expressed in human plaques compared with nonatherosclerotic mammary arteries and colocalized with splicing factor SC-35. Cell culture experiments indicated that p43 expression was associated with enhanced protein translation. On initiation of apoptosis or necrosis, p43 was cleaved by calpains and released as truncated protein p43(apoptosis-released factor [ARF]). Processing of p43 into endothelial monocyte activating polypeptide II was not observed. Full-length p43, but not p43(ARF) or endothelial monocyte activating polypeptide II, activated THP1 monocytes (upregulation of tumor necrosis factor alpha, interleukin 1 beta, interleukin 8, macrophage inflammatory protein (MIP)-1 alpha, MIP1 beta, MIP2 alpha) and endothelial cells (enhanced synthesis of E-selectin, vascular cell adhesion molecule-1, intercellular adhesion molecule-1, tissue factor). The chemotactic activity of p43 or fragments thereof was poor compared with ATP. Treatment of smooth muscle cells with p43 did not induce cell death. CONCLUSIONS: p43 is cleaved during apoptosis by calpains and released as a truncated protein that is harmless for the structure of the plaque.

Effect of statins on the viability of macrophages and smooth muscle cells.

Because macrophages play an important role in the destabilization of atherosclerotic plaques, and smooth muscle cells (SMCs) contribute to plaque stability, selective clearance of macrophages in atherosclerotic plaques is a promising strategy for plaque stabilization. In the present study, we investigated the effects of fluvastatin, simvastatin, lovastatin, and pravastatin on the viability of macrophages and SMCs. All statins, except pravastatin, induced cell death of J774A.1 macrophages after 24 hours, albeit with different sensitivity. The viability of rabbit aortic SMCs was hardly affected. Fluvastatin-induced macrophage cell death was characterized as apoptosis and could be reversed by isoprenoid intermediates of the mevalonate pathway. Peritoneal macrophages from male or female mice were much more resistant to statin-induced cell death. The high sensitivity of J774A.1 macrophages to statin-induced cell death was related to the strong 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in these cells. Macrophage and SMC content of rabbit atherosclerotic plaques was not affected after in vitro treatment with fluvastatin or lovastatin for 3 days. In conclusion, fluvastatin, simvastatin, and lovastatin, but not pravastatin, can selectively induce apoptosis in J774A.1 macrophages, but not in SMCs, primary macrophages or rabbit atherosclerotic plaques. This effect was related to the degree of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in the different cell types.