Association of Circulating Autophagy Proteins ATG5 and Beclin 1 with Acute Myocardial Infarction in a Case-Control Study.

INTRODUCTION: Acute myocardial infarction (AMI) is a main contributor of sudden cardiac death worldwide. The discovery of new biomarkers that can improve AMI risk prediction meets a major clinical need for the identification of high-risk patients and the tailoring of medical treatment. Previously, we reported that autophagy a highly conserved catabolic mechanism for intracellular degradation of cellular components is involved in atherosclerotic plaque phenotype and cardiac pathological remodeling. The crucial role of autophagy in the normal and diseased heart has been well described, and its activation functions as a pro-survival process in response to myocardial ischemia. However, autophagy is dysregulated in ischemia/reperfusion injury, thus promoting necrotic or apoptotic cardiac cell death. Very few studies have focused on the plasma levels of autophagy markers in cardiovascular disease patients, even though they could be companion biomarkers of AMI injury. The aims of the present study were to evaluate (1) whether variations in plasma levels of two key autophagy regulators autophagy-related gene 5 (ATG5) and Beclin 1 (the mammalian yeast ortholog Atg6/Vps30) are associated with AMI and (2) their potential for predicting AMI risk. METHODS: The case-control study population included AMI patients (n = 100) and control subjects (n = 99) at high cardiovascular risk but without known coronary disease. Plasma levels of ATG5 and Beclin 1 were measured in the whole population study by enzyme-linked immunosorbent assay. RESULTS: Multivariate analyses adjusted on common cardiovascular factors and medical treatments, and receiver operating characteristic curves demonstrated that ATG5 and Beclin 1 levels were inversely associated with AMI and provided original biomarkers for AMI risk prediction. CONCLUSION: Plasma levels of autophagy regulators ATG5 and Beclin 1 represent relevant candidate biomarkers associated with AMI.

Design and Synthesis of 1,3-Diarylpyrazoles and Investigation of Their Cytotoxicity and Antiparasitic Profile.

Herein, we report a series of 1,3-diarylpyrazoles that are analogues of compound 26/HIT 8. We previously identified this molecule as a 'hit' during a high-throughput screening campaign for autophagy inducers. A variety of synthetic strategies were utilized to modify the 1,3-diarylpyrazole core at its 1-, 3-, and 4-position. Compounds were assessed in vitro to identify their cytotoxicity properties. Of note, several compounds in the series displayed relevant cytotoxicity, which warrants scrutiny while interpreting biological activities that have been reported for structurally related molecules. In addition, antiparasitic activities were recorded against a range of human-infective protozoa, including Trypanosoma cruzi, T. brucei rhodesiense, and Leishmania infantum. The most interesting compounds displayed low micromolar whole-cell potencies against individual or several parasitic species, while lacking cytotoxicity against human cells.

Effect of erythrophagocytosis-induced ferroptosis during angiogenesis in atherosclerotic plaques.

Intraplaque (IP) angiogenesis is a key feature of advanced atherosclerotic plaques. Because IP vessels are fragile and leaky, erythrocytes are released and phagocytosed by macrophages (erythrophagocytosis), which leads to high intracellular iron content, lipid peroxidation and cell death. In vitro experiments showed that erythrophagocytosis by macrophages induced non-canonical ferroptosis, an emerging type of regulated necrosis that may contribute to plaque destabilization. Erythrophagocytosis-induced ferroptosis was accompanied by increased expression of heme-oxygenase 1 and ferritin, and could be blocked by co-treatment with third generation ferroptosis inhibitor UAMC-3203. Both heme-oxygenase 1 and ferritin were also expressed in erythrocyte-rich regions of carotid plaques from ApoE-/- Fbn1C1039G+/- mice, a model of advanced atherosclerosis with IP angiogenesis. The effect of UAMC-3203 (12.35 mg/kg/day) on atherosclerosis was evaluated in ApoE-/- Fbn1C1039G+/- mice fed a western-type diet (WD) for 12 weeks (n = 13 mice/group) or 20 weeks (n = 16-21 mice/group) to distinguish between plaques without and with established IP angiogenesis, respectively. A significant decrease in carotid plaque thickness was observed after 20 weeks WD (87 ± 19 µm vs. 166 ± 20 µm, p = 0.006), particularly in plaques with confirmed IP angiogenesis or hemorrhage (108 ± 35 µm vs. 322 ± 40 µm, p = 0.004). This effect was accompanied by decreased IP heme-oxygenase 1 and ferritin expression. UAMC-3203 did not affect carotid plaques after 12 weeks WD or plaques in the aorta, which typically do not develop IP angiogenesis. Altogether, erythrophagocytosis-induced ferroptosis during IP angiogenesis leads to larger atherosclerotic plaques, an effect that can be prevented by ferroptosis inhibitor UAMC-3203.

Regulated Necrosis in Atherosclerosis.

During atherosclerosis, lipid-rich plaques are formed in large- and medium-sized arteries, which can reduce blood flow to tissues. This situation becomes particularly precarious when a plaque develops an unstable phenotype and becomes prone to rupture. Despite advances in identifying and treating vulnerable plaques, the mortality rate and disability caused by such lesions remains the number one health threat in developed countries. Vulnerable, unstable plaques are characterized by a large necrotic core, implying a prominent role for necrotic cell death in atherosclerosis and plaque destabilization. Necrosis can occur accidentally or can be induced by tightly regulated pathways. Over the past decades, different forms of regulated necrosis, including necroptosis, ferroptosis, pyroptosis, and secondary necrosis, have been identified, and these may play an important role during atherogenesis. In this review, we describe several forms of necrosis that may occur in atherosclerosis and how pharmacological modulation of these pathways can stabilize vulnerable plaques. Moreover, some challenges of targeting necrosis in atherosclerosis such as the presence of multiple death-inducing stimuli in plaques and extensive cross-talk between necrosis pathways are discussed. A better understanding of the role of (regulated) necrosis in atherosclerosis and the mechanisms contributing to plaque destabilization may open doors to novel pharmacological strategies and will enable clinicians to tackle the residual cardiovascular risk that remains in many atherosclerosis patients.

PFKFB3 gene deletion in endothelial cells inhibits intraplaque angiogenesis and lesion formation in a murine model of venous bypass grafting.

Vein grafting is a frequently used surgical intervention for cardiac revascularization. However, vein grafts display regions with intraplaque (IP) angiogenesis, which promotes atherogenesis and formation of unstable plaques. Graft neovessels are mainly composed of endothelial cells (ECs) that largely depend on glycolysis for migration and proliferation. In the present study, we aimed to investigate whether loss of the glycolytic flux enzyme phosphofructokinase-2/fructose-2,6-bisphosphatase 3 (PFKFB3) in ECs inhibits IP angiogenesis and as such prevents unstable plaque formation. To this end, apolipoprotein E deficient (ApoE-/-) mice were backcrossed to a previously generated PFKFB3fl/fl Cdh5iCre mouse strain. Animals were injected with either corn oil (ApoE-/-PFKFB3fl/fl) or tamoxifen (ApoE-/-PFKFB3ECKO), and were fed a western-type diet for 4 weeks prior to vein grafting. Hereafter, mice received a western diet for an additional 28 days and were then sacrificed for graft assessment. Size and thickness of vein graft lesions decreased by 35 and 32%, respectively, in ApoE-/-PFKFB3ECKO mice compared to controls, while stenosis diminished by 23%. Moreover, vein graft lesions in ApoE-/-PFKFB3ECKO mice showed a significant reduction in macrophage infiltration (29%), number of neovessels (62%), and hemorrhages (86%). EC-specific PFKFB3 deletion did not show obvious adverse effects or changes in general metabolism. Interestingly, RT-PCR showed an increased M2 macrophage signature in vein grafts from ApoE-/-PFKFB3ECKO mice. Altogether, EC-specific PFKFB3 gene deletion leads to a significant reduction in lesion size, IP angiogenesis, and hemorrhagic complications in vein grafts. This study demonstrates that inhibition of endothelial glycolysis is a promising therapeutic strategy to slow down plaque progression.

Partial Inhibition of Glycolysis Reduces Atherogenesis Independent of Intraplaque Neovascularization in Mice.

OBJECTIVE: Intraplaque neovascularization is an important feature of unstable human atherosclerotic plaques. However, its impact on plaque formation and stability is poorly studied. Because proliferating endothelial cells generate up to 85% of their ATP from glycolysis, we investigated whether pharmacological inhibition of glycolytic flux by the small-molecule 3PO (3-[3-pyridinyl]-1-[4-pyridinyl]-2-propen-1-one) could have beneficial effects on plaque formation and composition. Approach and Results: ApoE-/- (apolipoprotein E deficient) mice treated with 3PO (50 µg/g, ip; 4×/wk, 4 weeks) showed a metabolic switch toward ketone body formation. Treatment of ApoE-/-Fbn1C1039G+/- mice with 3PO (50 µg/g, ip) either after 4 (preventive, twice/wk, 10 weeks) or 16 weeks of Western diet (curative, 4×/wk, 4 weeks) inhibited intraplaque neovascularization by 50% and 38%, respectively. Plaque formation was significantly reduced in all 3PO-treated animals. This effect was independent of intraplaque neovascularization. In vitro experiments showed that 3PO favors an anti-inflammatory M2 macrophage subtype and suppresses an M1 proinflammatory phenotype. Moreover, 3PO induced autophagy, which in turn impaired NF-κB (nuclear factor-kappa B) signaling and inhibited TNF-α (tumor necrosis factor-alpha)-mediated VCAM-1 (vascular cell adhesion molecule-1) and ICAM-1 (intercellular adhesion molecule-1) upregulation. Consistently, a preventive 3PO regimen reduced endothelial VCAM-1 expression in vivo. Furthermore, 3PO improved cardiac function in ApoE-/-Fbn1C1039G+/- mice after 10 weeks of treatment. CONCLUSIONS: Partial inhibition of glycolysis restrained intraplaque angiogenesis without affecting plaque composition. However, less plaques were formed, which was accompanied by downregulation of endothelial adhesion molecules-an event that depends on autophagy induction. Inhibition of coronary plaque formation by 3PO resulted in an overall improved cardiac function.

The PFKFB3 Inhibitor AZ67 Inhibits Angiogenesis Independently of Glycolysis Inhibition.

Angiogenesis is the process of new blood vessel formation. In this complex orchestrated growth, many factors are included. Lately, focus has shifted to endothelial cell metabolism, particularly to the PFKFB3 protein, a key regulatory enzyme of the glycolytic pathway. A variety of inhibitors of this important target have been studied, and a plethora of biological effects related to the process of angiogenesis have been reported. However, recent studies have disputed their mechanism of action, questioning whether all the effects are indeed due to PFKFB3 inhibition. Remarkably, the most well-studied inhibitor, 3PO, does not bind to PFKFB3, raising questions about this target. In our study, we aimed to elucidate the effects of PFKFB3 inhibition in angiogenesis by using the small molecule AZ67. We used isothermal titration calorimetry and confirmed binding to PFKFB3. In vitro, AZ67 did not decrease lactate production in endothelial cells (ECs), nor ATP levels, but exhibited good inhibitory efficacy in the tube-formation assay. Surprisingly, this was independent of EC migratory and proliferative abilities, as this was not diminished upon treatment. Strikingly however, even the lowest dose of AZ67 demonstrated significant inhibition of angiogenesis in vivo. To our knowledge, this is the first study to demonstrate that the process of angiogenesis can be disrupted by targeting PFKFB3 independently of glycolysis inhibition.

Acetylsalicylic Acid Reduces Passive Aortic Wall Stiffness and Cardiovascular Remodelling in a Mouse Model of Advanced Atherosclerosis.

Acetylsalicylic acid (ASA) is widely used in secondary prevention of cardiovascular (CV) disease, mainly because of its antithrombotic effects. Here, we investigated whether ASA can prevent the progression of vessel wall remodelling, atherosclerosis, and CV complications in apolipoprotein E deficient (ApoE-/-) mice, a model of stable atherosclerosis, and in ApoE-/- mice with a mutation in the fibrillin-1 gene (Fbn1C1039G+/-), which is a model of elastic fibre fragmentation, accompanied by exacerbated unstable atherosclerosis. Female ApoE-/- and ApoE-/-Fbn1C1039G+/- mice were fed a Western diet (WD). At 10 weeks of WD, the mice were randomly divided into four groups, receiving either ASA 5 mg/kg/day in the drinking water (ApoE-/- (n = 14), ApoE-/-Fbn1C1039G+/- (n = 19)) or plain drinking water (ApoE-/- (n = 15), ApoE-/-Fbn1C1039G+/- (n = 21)) for 15 weeks. ApoE-/-Fbn1C1039G+/- mice showed an increased neutrophil-lymphocyte ratio (NLR) compared to ApoE-/- mice, and this effect was normalised by ASA. In the proximal ascending aorta wall, ASA-treated ApoE-/-Fbn1C1039G+/- mice showed less p-SMAD2/3 positive nuclei, a lower collagen percentage and an increased elastin/collagen ratio, consistent with the values measured in ApoE-/- mice. ASA did not affect plaque progression, incidence of myocardial infarction and survival of ApoE-/-Fbn1C1039G+/- mice, but systolic blood pressure, cardiac fibrosis and hypertrophy were reduced. In conclusion, ASA normalises the NLR, passive wall stiffness and cardiac remodelling in ApoE-/-Fbn1C1039G+/- mice to levels observed in ApoE-/- mice, indicating additional therapeutic benefits of ASA beyond its classical use.

Serum Corticosterone and Insulin Resistance as Early Biomarkers in the hAPP23 Overexpressing Mouse Model of Alzheimer's Disease.

Increasing epidemiological evidence highlights the association between systemic insulin resistance and Alzheimer's disease (AD). As insulin resistance can be caused by high-stress hormone levels and since hypercortisolism appears to be an important risk factor of AD, we aimed to investigate the systemic insulin functionality and circulating stress hormone levels in a mutant humanized amyloid precursor protein (APP) overexpressing (hAPP23+/-) AD mouse model. Memory and spatial learning of male hAPP23+/- and C57BL/6 (wild type, WT) mice were assessed by a Morris Water Maze (MWM) test at the age of 4 and 12 months. The systemic metabolism was examined by intraperitoneal glucose and insulin tolerance tests (GTT, ITT). Insulin and corticosterone levels were determined in serum. In the hippocampus, parietal and occipital cortex of hAPP23+/- brains, amyloid-beta (Aß) deposits were present at 12 months of age. MWM demonstrated a cognitive decline in hAPP23+/- mice at 12 but not at 4 months, evidenced by increasing total path lengths and deteriorating probe trials compared to WT mice. hAPP23+/- animals presented increased serum corticosterone levels compared to WT mice at both 4 and 12 months. hAPP23+/- mice exhibited peripheral insulin resistance compared to WT mice at 4 months, which stabilized at 12 months of age. Serum insulin levels were similar between genotypes at 4 months of age but were significantly higher in hAPP23+/- mice at 12 months of age. Peripheral glucose homeostasis remained unchanged. These results indicate that peripheral insulin resistance combined with elevated circulating stress hormone levels could be potential biomarkers of the pre-symptomatic phase of AD.

Doxorubicin Impairs Smooth Muscle Cell Contraction: Novel Insights in Vascular Toxicity.

Clinical and animal studies have demonstrated that chemotherapeutic doxorubicin (DOX) increases arterial stiffness, a predictor of cardiovascular risk. Despite consensus about DOX-impaired endothelium-dependent vasodilation as a contributing mechanism, some studies have reported conflicting results on vascular smooth muscle cell (VSMC) function after DOX treatment. The present study aimed to investigate the effects of DOX on VSMC function. To this end, mice received a single injection of 4 mg DOX/kg, or mouse aortic segments were treated ex vivo with 1 µM DOX, followed by vascular reactivity evaluation 16 h later. Phenylephrine (PE)-induced VSMC contraction was decreased after DOX treatment. DOX did not affect the transient PE contraction dependent on Ca2+ release from the sarcoplasmic reticulum (0 mM Ca2+), but it reduced the subsequent tonic phase characterised by Ca2+ influx. These findings were supported by similar angiotensin II and attenuated endothelin-1 contractions. The involvement of voltage-gated Ca2+ channels in DOX-decreased contraction was excluded by using levcromakalim and diltiazem in PE-induced contraction and corroborated by similar K+ and serotonin contractions. Despite the evaluation of multiple blockers of transient receptor potential channels, the exact mechanism for DOX-decreased VSMC contraction remains elusive. Surprisingly, DOX reduced ex vivo but not in vivo arterial stiffness, highlighting the importance of appropriate timing for evaluating arterial stiffness in DOX-treated patients.

Defective autophagy in vascular smooth muscle cells increases passive stiffness of the mouse aortic vessel wall.

Aging and associated progressive arterial stiffening are both important predictors for the development of cardiovascular diseases. Recent evidence showed that autophagy, a catabolic cellular mechanism responsible for nutrient recycling, plays a major role in the physiology of vascular cells such as endothelial cells and vascular smooth muscle cells (VSMCs). Moreover, several autophagy inducing compounds are effective in treating arterial stiffness. Yet, a direct link between VSMC autophagy and arterial stiffness remains largely unidentified. Therefore, we investigated the effects of a VSMC-specific deletion of the essential autophagy-related gene Atg7 in young mice (3.5 months) (Atg7F/F SM22α-Cre+ mice) on the biomechanical properties of the aorta, using an in-house developed Rodent Oscillatory Tension Set-up to study Arterial Compliance (ROTSAC). Aortic segments of Atg7F/F SM22α-Cre+ mice displayed attenuated compliance and higher arterial stiffness, which was more evident at higher distention pressures. Passive aortic wall remodeling, rather than differences in VSMC tone, is responsible for these phenomena, since differences in compliance and stiffness between Atg7+/+ SM22α-Cre+ and Atg7F/F SM22α-Cre+ aortas were more pronounced when VSMCs were completely relaxed by the addition of exogenous nitric oxide. These observations are supported by histological data showing a 13% increase in medial wall thickness and a 14% decrease in elastin along with elevated elastin fragmentation. In addition, expression of the calcium-binding protein S100A4, which is linked to matrix remodeling, was elevated in aortic segments of Atg7F/F SM22α-Cre+ mice. Overall, these findings illustrate that autophagy exerts a crucial role in defining arterial wall compliance.

Three-Dimensional Imaging of Intraplaque Neovascularization in a Mouse Model of Advanced Atherosclerosis.

Multiple lines of evidence suggest that intraplaque (IP) neovascularization promotes atherosclerotic plaque growth, destabilization, and rupture. However, pharmacological inhibition of IP neovascularization remains largely unexplored due to the limited number of animal models that develop IP neovessels and the lack of reliable methods for visualizing IP angiogenesis. Here, we applied 3D confocal microscopy with an optimized tissue-clearing process, immunolabeling-enabled three-dimensional imaging of solvent-cleared organs, to visualize IP neovessels in apolipoprotein E-deficient (ApoE-/-) mice carrying a heterozygous mutation (C1039+/-) in the fibrillin-1 gene. Unlike regular ApoE-/- mice, this mouse model is characterized by the presence of advanced plaques with evident IP neovascularization. Plaques were stained with antibodies against endothelial marker CD31 for 3 days, followed by incubation with fluorescently labeled secondary antibodies. Subsequent tissue clearing with dichloromethane (DCM)/methanol, DCM, and dibenzyl ether allowed easy visualization and 3D reconstruction of the IP vascular network while plaque morphology remained intact.

Autophagy Dynamics and Modulation in a Rat Model of Renal Ischemia-Reperfusion Injury.

Renal ischemia-reperfusion (IR) injury leading to cell death is a major cause of acute kidney injury, contributing to morbidity and mortality. Autophagy counteracts cell death by removing damaged macromolecules and organelles, making it an interesting anchor point for treatment strategies. However, autophagy is also suggested to enhance cell death when the ischemic burden is too strong. To investigate whether the role of autophagy depends on the severity of ischemic stress, we analyzed the dynamics of autophagy and apoptosis in an IR rat model with mild (45 min) or severe (60 min) renal ischemia. Following mild IR, renal injury was associated with reduced autophagy, enhanced mammalian target of rapamycin (mTOR) activity, and apoptosis. Severe IR, on the other hand, was associated with a higher autophagic activity, independent of mTOR, and without affecting apoptosis. Autophagy stimulation by trehalose injected 24 and 48 h prior to onset of severe ischemia did not reduce renal injury markers nor function, but reduced apoptosis and restored tubular dilation 7 days post reperfusion. This suggests that trehalose-dependent autophagy stimulation enhances tissue repair following an IR injury. Our data show that autophagy dynamics are strongly dependent on the severity of IR and that trehalose shows the potential to trigger autophagy-dependent repair processes following renal IR injury.

The Protective Effects of the Autophagic and Lysosomal Machinery in Vascular and Valvular Calcification: A Systematic Review.

BACKGROUND: Autophagy is a highly conserved catabolic homeostatic process, crucial for cell survival. It has been shown that autophagy can modulate different cardiovascular pathologies, including vascular calcification (VCN). OBJECTIVE: To assess how modulation of autophagy, either through induction or inhibition, affects vascular and valvular calcification and to determine the therapeutic applicability of inducing autophagy. DATA SOURCES: A systematic review of English language articles using MEDLINE/PubMed, Web of Science (WoS) and the Cochrane library. The search terms included autophagy, autolysosome, mitophagy, endoplasmic reticulum (ER)-phagy, lysosomal, calcification and calcinosis. Study characteristics: Thirty-seven articles were selected based on pre-defined eligibility criteria. Thirty-three studies (89%) studied vascular smooth muscle cell (VSMC) calcification of which 27 (82%) studies investigated autophagy and six (18%) studies lysosomal function in VCN. Four studies (11%) studied aortic valve calcification (AVCN). Thirty-four studies were published in the time period 2015-2020 (92%). CONCLUSION: There is compelling evidence that both autophagy and lysosomal function are critical regulators of VCN, which opens new perspectives for treatment strategies. However, there are still challenges to overcome, such as the development of more selective pharmacological agents and standardization of methods to measure autophagic flux.

Synthesis and evaluation of novel benzotropolones as Atg4B inhibiting autophagy blockers.

Autophagy is an intracellular degradation/recycling pathway that provides nutrients and building blocks to cellular metabolism and keeps the cytoplasm clear of obsolete proteins and organelles. During recent years, dysregulated autophagy activity has been reported to be a characteristic of many different disease types, including cancer and neurodegenerative disorders. This has created a strong case for development of autophagy modulating compounds as potential treatments for these diseases. Inhibitors of autophagy have been proposed as a therapeutic intervention in, e.g., advanced cancer, and inhibiting the cysteine protease Atg4B has been put forward as a main strategy to block autophagy. We recently identified and demonstrated -both in vitro and in vivo - that compounds with a benzotropolone basic structure targeting Atg4B, can significantly slow down tumor growth and potentiate the effect of classical chemotherapy. In this study we report the synthesis and inhibition profile of new benzotropolone derivatives with additional structural modifications at 6 different positions. To obtain a solid inhibition profile, all compounds were evaluated on three levels, including two cell-based assays to confirm autophagy and intracellular Atg4B inhibition and an SDS-PAGE-based experiment to assess in vitro Atg4B affinity. Several molecules with a promising profile were identified.

Cytoprotective effects of transgenic neuroglobin overexpression in an acute and chronic mouse model of ischemic heart disease.

Neuroglobin (NGB) is an oxygen-binding protein that is mainly expressed in nervous tissues where it is considered to be neuroprotective during ischemic brain injury. Interestingly, transgenic mice overexpressing NGB reveal cytoprotective effects on tissues lacking endogenous NGB, which might indicate a therapeutic role for NGB in a broad range of ischemic conditions. In the present study, we investigated the effect of NGB overexpression on survival as well as on the size and occurrence of myocardial infarctions (MI) in a mouse model of acute MI (AMI) and a model of advanced atherosclerosis (ApoE -/- Fbn1 C1039G+/- mice), in which coronary plaques and MI develop in mice being fed a Western-type diet. Overexpression of NGB significantly enhanced post-AMI survival and reduced MI size by 14% 1 week after AMI. Gene expression analysis of the infarction border showed reduction of tissue hypoxia and attenuation of hypoxia-induced inflammatory pathways, which might be responsible for these beneficial effects. In contrast, NGB overexpression did not affect survival or occurrence of MI in the atherosclerotic mice although the incidence of coronary plaques was significantly reduced. In conclusion, NGB proved to act cytoprotectively during MI in the acute setting while this effect was less pronounced in the atherosclerosis model.

Autophagy in vascular disease.

Autophagy is a reparative, life-sustaining process by which cytoplasmic components are sequestered in double-membrane vesicles and degraded on fusion with lysosomal compartments. Growing evidence reveals that basal autophagy is an essential in vivo process mediating proper vascular function. Moreover, autophagy is stimulated by many stress-related stimuli in the arterial wall to protect endothelial cells and smooth muscle cells against cell death and the initiation of vascular disease, in particular atherosclerosis. Basal autophagy is atheroprotective during early atherosclerosis but becomes dysfunctional in advanced atherosclerotic plaques. Little is known about autophagy in other vascular disorders, such as aneurysm formation, arterial aging, vascular stiffness, and chronic venous disease, even though autophagy is often impaired. This finding highlights the need for pharmacological interventions with compounds that stimulate the prosurvival effects of autophagy in the vasculature. A large number of animal studies and clinical trials have indicated that oral or stent-based delivery of the autophagy inducer rapamycin or derivatives thereof, collectively known as rapalogs, effectively inhibit the basic mechanisms that control growth and destabilization of atherosclerotic plaques. Other autophagy-inducing drugs, such as spermidine or add-on therapy with widely used antiatherogenic compounds, including statins and metformin, are potentially useful to prevent vascular disease with minimal adverse effects.

A novel set-up for the ex vivo analysis of mechanical properties of mouse aortic segments stretched at physiological pressure and frequency.

KEY POINTS: Cyclic stretch is known to alter intracellular pathways involved in vessel tone regulation. We developed a novel set-up that allows straightforward characterization of the biomechanical properties of the mouse aorta while stretched at a physiological heart rate (600 beats min-1 ). Active vessel tone was shown to have surprisingly large effects on isobaric stiffness. The effect of structural vessel wall alterations was confirmed using a genetic mouse model. This set-up will contribute to a better understanding of how active vessel wall components and mechanical stimuli such as stretch frequency and amplitude regulate aortic mechanics. ABSTRACT: 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-1 . 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 (EP ; 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, EP 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.

Hepatocellular autophagy modulates the unfolded protein response and fasting-induced steatosis in mice.

Autophagy and the unfolded protein response (UPR) are key cellular homeostatic mechanisms and are both involved in liver diseases, including nonalcoholic fatty liver disease (NAFLD). Although increasing but conflicting results link these mechanisms to lipid metabolism, their role and potential cross talk herein have been poorly investigated. Therefore, we assessed the effects of hepatocyte-specific autophagy deficiency on liver parenchyma, the UPR, and lipid metabolism. Adult hepatocellular-specific autophagy-deficient mice (Atg7F/FAlb-Cre+) were compared with their autophagy-competent littermates (Atg7+/+Alb-Cre+). Livers were analyzed by electron microscopy, histology, real-time qPCR, and Western blotting. Atg7F/FAlb-Cre+ mice developed hepatomegaly with significant parenchymal injury, as shown by inflammatory infiltrates, hepatocellular apoptosis, pericellular fibrosis, and a pronounced ductular reaction. Surprisingly, the UPR exhibited a pathway-selective pattern upon autophagy deficiency. The activity of the adaptive activating transcription factor 6 (ATF6) pathway was abolished, whereas the proapoptotic protein kinase RNA-like ER kinase pathway was increased compared with Atg7+/+Alb-Cre+ mice. The inositol-requiring enzyme-1α signal was unaltered. Fasting-induced steatosis was absent in Atg7F/FAlb-Cre+ mice. Remarkably, some isolated islands of fat-containing and autophagy-competent cells were observed in these livers. Hepatocellular autophagy is essential for parenchymal integrity in mice. Moreover, in the case of autophagy deficiency, the three different UPR branches are pathway selectively modulated. Attenuation of the ATF6 pathway might explain the observed impairment of fasting-induced steatosis. Finally, autophagy and lipid droplets are directly linked to each other.

Potential therapeutic effects of mTOR inhibition in atherosclerosis.

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.