A utophagy is a complex intracellular process that delivers cytoplasmic constituents for degradation into lysosomes.1,2 Three main types of autophagy have been described: (1) microautophagy, comprising direct engulfment of cytoplasmic material by lysosomes via inward invaginations of the lysosomal membrane, (2) macroautophagy, characterized by formation of double-membrane sequestering compartments termed autophagosomes that fuse with lysosomes for delivery of cytoplasmic cargo, and (3) chaperone-mediated autophagy, mediated by a chaperone complex and lysosomal-associated membrane protein type 2A to degrade cytosolic proteins with a specific targeting motif. The term autophagy usually refers to macroautophagy, which is the most prevalent and beststudied form of autophagy. Also in this review, we will focus exclusively on macroautophagy, further cited as autophagy.Autophagy occurs at basal levels in most tissues to allow constitutive turnover of cytosolic components but is stimulated by environmental stress-related signals (eg, nutrient deprivation and oxidative injury) to recycle nutrients and to generate energy for maintenance of cell viability in unfavorable conditions.1 In addition to cellular stress, basal autophagy can be intensified by specific drugs, 3 indicating that the autophagic machinery is a potential therapeutic target for diverse diseases. Indeed, given that autophagy is involved in the prevention

Despite significant improvement in the management of atherosclerosis, this slowly progressing disease continues to affect countless patients around the world. Recently, the mechanistic target of rapamycin (mTOR) has been identified as a pre-eminent factor in the development of atherosclerosis. mTOR is a constitutively active kinase found in two different multiprotein complexes, mTORC1 and mTORC2. Pharmacological interventions with a class of macrolide immunosuppressive drugs, called rapalogs, have shown undeniable evidence of the value of mTORC1 inhibition to prevent the development of atherosclerotic plaques in several animal models. Rapalog-eluting stents have also shown extraordinary results in humans, even though the exact mechanism for this anti-atherosclerotic effect remains elusive. Unfortunately, rapalogs are known to trigger diverse undesirable effects owing to mTORC1 resistance or mTORC2 inhibition. These adverse effects include dyslipidaemia and insulin resistance, both known triggers of atherosclerosis. Several strategies, such as combination therapy with statins and metformin, have been suggested to oppose rapalog-mediated adverse effects. Statins and metformin are known to inhibit mTORC1 indirectly via 5' adenosine monophosphate-activated protein kinase (AMPK) activation and may hold the key to exploit the full potential of mTORC1 inhibition in the treatment of atherosclerosis. Intermittent regimens and dose reduction have also been proposed to improve rapalog's mTORC1 selectivity, thereby reducing mTORC2-related side effects.

Spermidine inhibits lipid accumulation and necrotic core formation through stimulation of cholesterol efflux, albeit without changing plaque size or cellular composition. These effects, which are driven by autophagy in VSMCs, support the general idea that autophagy induction is potentially useful to prevent vascular disease.

Cyclic stretch is a major contributor to vascular function. However, isolated mouse aortas are frequently studied at low stretch frequency or even in isometric conditions. Pacing experiments in rodents and humans show that arterial compliance is stretch frequency dependent. The Rodent Oscillatory Tension Set-up to study Arterial Compliance is an in-house developed organ bath set-up that clamps aortic segments to imposed preloads at physiological rates up to 600 beats min . The technique enables us to derive pressure-diameter loops and assess biomechanical properties of the segment. To validate the applicability of this set-up we aimed to confirm the effects of distension pressure and vascular smooth muscle tone on arterial stiffness. At physiological stretch frequency (10 Hz), the Peterson modulus (E ; 293 (10) mmHg) for wild-type mouse aorta increased 22% upon a rise in pressure from 80-120 mmHg to 100-140 mmHg, while, at normal pressure, E increased 80% upon maximal contraction of the vascular smooth muscle cells. We further validated the method using a mouse model with a mutation in the fibrillin-1 gene and an endothelial nitric oxide synthase knock-out model. Both models are known to have increased arterial stiffness, and this was confirmed using the set-up. To our knowledge, this is the first set-up that facilitates the study of biomechanical properties of mouse aortic segments at physiological stretch frequency and pressure. We believe that this set-up can contribute to a better understanding of how cyclic stretch frequency, amplitude and active vessel wall components influence arterial stiffening.

Inhibitors of the mechanistic target of rapamycin (mTOR) have unique antiatherosclerotic effects, such as depletion of plaque macrophages, induction of autophagy, and activation of cholesterol efflux. However, a common side effect of their use is dyslipidemia, a well-known risk factor for atherosclerosis. Indeed, mTOR inhibitors prevent lipid storage, increase low-density lipoprotein cholesterol levels, and activate lipolysis. Although the net effect of mTOR inhibition seems favorable, the use of cholesterol lowering drugs to manage dyslipidemia remains the most recommended strategy.

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BACKGROUND AND PURPOSEEverolimus is an allosteric inhibitor of the mechanistic target of rapamycin complex 1 (mTORC1) widely known for its potent autophagy stimulating properties. Because everolimus shows poor solubility and stability in aqueous solutions, long-term in vivo administration in preclinical models is challenging. The aim of the present study was to evaluate the effects of short-term and long-term everolimus administration on mTORC1 inhibition and autophagy induction in mice. EXPERIMENTAL APPROACHWe developed a vehicle in which everolimus was solubilized and stable at 37°C for at least 1 month. Using osmotic minipumps, GFP microtubule-associated protein light chain 3 transgenic mice were treated continuously either with vehicle or everolimus (1.5 mg·kg À1 per day) for 3 or 28 days. Alternatively, a regimen consisting of intermittent everolimus administration (every other day) for 56 days by oral gavage was used. Autophagy markers and mTORC1 activation status were investigated in the liver. KEY RESULTSAs expected, everolimus inhibited mTORC1 and stimulated autophagy in the liver after 3 days of treatment. However, continuous administration for 28 days resulted in hyperactivation of the Akt1-mTORC1 pathway accompanied by a remarkable decrease in autophagy markers. Everolimus given intermittently for 56 days partially rescued mTORC1 sensitivity to the drug but without inducing autophagy. The failure to induce autophagy following long-term everolimus administration was due to uncoupling of the mTORC1 substrate unc-51 like autophagy activating kinase 1. CONCLUSIONS AND IMPLICATIONSOur data encourage the use of intermittent everolimus regimens to prevent tolerance and to extend its activity. AbbreviationsACTB, β-actin; LC3, microtubule-associated protein light chain 3; mTORC1/2, mechanistic target of rapamycin, complex 1/2; RCC, renal cell carcinoma; p70S6K, ribosomal protein S6 kinase B1; S6rp, S6 ribosomal protein; SQSTM1, sequestosome 1; ULK1, unc-51 like autophagy activating kinase

Background and aims: Inhibition of the mechanistic target of rapamycin (mTOR) is a promising approach to halt atherogenesis in different animal models. This study evaluated whether the mTOR inhibitor everolimus can stabilize pre-existing plaques, prevent cardiovascular complications and improve survival in a mouse model of advanced atherosclerosis. Methods: ApoE-/-Fbn1 C1039G+/mice (n=24) were fed a Western diet (WD) for 12 weeks. Subsequently, mice were treated with everolimus (1.5 mg/kg daily) or vehicle for another 12 weeks while the WD continued. Results: Despite hypercholesterolemia, everolimus treatment was associated with a reduction in circulating Ly6C high monocytes (15 vs. 28% of total leukocytes, p=0.046), a depletion of plaque macrophages (2.1 vs. 4.1%, p=0.040) and an abolishment of intraplaque neovascularization, which are all indicative of a more stable plaque phenotype. Moreover, everolimus reduced hypoxic brain damage and improved cardiac function, which led to increased survival (100 vs. 67% of animals, p=0.038). Conclusions: Everolimus enhances features of plaque stability and counters cardiovascular complications in ApoE-/-Fbn1 C1039G+/mice, even when administered at a later stage of the disease.