Role of autophagy in ischemic stroke: insights from animal models and preliminary evidence in the human disease.

Stroke represents a main cause of death and permanent disability worldwide. The molecular mechanisms underlying cerebral injury in response to the ischemic insults are not completely understood. In this article, we summarize recent evidence regarding the role of autophagy in the pathogenesis of ischemic stroke by reviewing data obtained in murine models of either transient or permanent middle cerebral artery occlusion, and in the stroke-prone spontaneously hypertensive rat. Few preliminary observational studies investigating the role of autophagy in subjects at high cerebrovascular risk and in cohorts of stroke patients were also reviewed. Autophagy plays a dual role in neuronal and vascular cells by exerting both protective and detrimental effects depending on its level, duration of stress and type of cells involved. Protective autophagy exerts adaptive mechanisms which reduce neuronal loss and promote survival. On the other hand, excessive activation of autophagy leads to neuronal cell death and increases brain injury. In conclusion, the evidence reviewed suggests that a proper manipulation of autophagy may represent an interesting strategy to either prevent or reduce brain ischemic injury.

Mitophagy modulation for the treatment of cardiovascular diseases.

BACKGROUND: Defects of mitophagy, the selective form of autophagy for mitochondria, are commonly observed in several cardiovascular diseases and represent the main cause of mitochondrial dysfunction. For this reason, mitophagy has emerged as a novel and potential therapeutic target. METHODS: In this review, we discuss current evidence about the biological significance of mitophagy in relevant preclinical models of cardiac and vascular diseases, such as heart failure, ischemia/reperfusion injury, metabolic cardiomyopathy and atherosclerosis. RESULTS: Multiple studies have shown that cardiac and vascular mitophagy is an adaptive mechanism in response to stress, contributing to cardiovascular homeostasis. Mitophagy defects lead to cell death, ultimately impairing cardiac and vascular function, whereas restoration of mitophagy by specific compounds delays disease progression. CONCLUSIONS: Despite previous efforts, the molecular mechanisms underlying mitophagy activation in response to stress are not fully characterized. A comprehensive understanding of different forms of mitophagy active in the cardiovascular system is extremely important for the development of new drugs targeting this process. Human studies evaluating mitophagy abnormalities in patients at high cardiovascular risk also represent a future challenge.

Molecular mechanisms of naringenin modulation of mitochondrial permeability transition acting on F1FO-ATPase and counteracting saline load-induced injury in SHRSP cerebral endothelial cells.

Naringenin (NRG) was characterized for its ability to counteract mitochondrial dysfunction which is linked to cardiovascular diseases. The F1FO-ATPase can act as a molecular target of NRG. The interaction of NRG with this enzyme can avoid the energy transmission mechanism of ATP hydrolysis, especially in the presence of Ca2+ cation used as cofactor. Indeed, NRG was a selective inhibitor of the hydrophilic F1 domain displaying a binding site overlapped with quercetin in the inside surface of an annulus made by the three α and the three ß subunits arranged alternatively in a hexamer. The kinetic constant of inhibition suggested that NRG preferred the enzyme activated by Ca2+ rather than the F1FO-ATPase activated by the natural cofactor Mg2+. From the inhibition type mechanism of NRG stemmed the possibility to speculate that NRG can prevent the activation of F1FO-ATPase by Ca2+. The event correlated to the protective role in the mitochondrial permeability transition pore opening by NRG as well as to the reduction of ROS production probably linked to the NRG chemical structure with antioxidant action. Moreover, in primary cerebral endothelial cells (ECs) obtained from stroke prone spontaneously hypertensive rats NRG had a protective effect on salt-induced injury by restoring cell viability and endothelial cell tube formation while also rescuing complex I activity.

miR-93 and synaptotagmin-7: two novel players in the regulation of autophagy during cardiac hypertrophy.

The molecular mechanisms involved in the transition of cardiac hypertrophy to heart failure (HF) are not fully characterized. Autophagy is a catabolic, self-renewal intracellular mechanism, which protects the heart during HF. In the heart of a mouse model of angiotensin-II-induced hypertrophy, Sun and colleagues demonstrated that reduced levels of miR-93 lead to synaptotagmin-7 (Syt-7) upregulation and consequent inhibition of autophagy. miR-93 overexpression or syt-7 inhibition rescues autophagy and maladaptive hypertrophy. This research identifies new players in the pathophysiology of cardiac hypertrophy, opening innovative therapeutic perspectives. miR-93 may also be considered in the future as a novel circulating biomarker for patients at high risk to develop HF.

Abnormal expression of sphingolipid-metabolizing enzymes in the heart of spontaneously hypertensive rat models.

Sphingolipids exert important roles within the cardiovascular system and related diseases. Perturbed sphingolipid metabolism was previously reported in cerebral and renal tissues of spontaneously hypertensive rats (SHR). Specific defects related to the synthesis of sphingolipids and to the metabolism of Sphingosine-1-Phospahte (S1P) were exclusively identified in the stroke-prone (SHRSP) with the respect to the stroke-resistant (SHRSR) strain. In this study, we explored any existing perturbation in either protein or gene expression of enzymes involved in the sphingolipid pathways in cardiac tissue from both SHRSP and SHRSR strains, compared to the normotensive Wistar Kyoto (WKY) strain. The two hypertensive rat models showed an overall perturbation of the expression of different enzymes involved in the sphingolipid metabolism in the heart. In particular, whereas the expression of the S1P-metabolizing-enzyme, SPHK2, was significantly reduced in both SHR strains, SGPL1 protein levels were decreased only in SHRSP. The protein levels of S1P receptors 1-3 were reduced only in the cardiac tissue of SHRSP, whereas S1PR2 levels were reduced in both SHR strains. The de novo synthesis of sphingolipids was aberrant in the two hypertensive strains. A significant reduction of mRNA expression of the Sgms1 and Smpd3 enzymes, implicated in the metabolism of sphingomyelin, was found in both hypertensive strains. Interestingly, Smpd2, devoted to sphingomyelin degradation, was reduced only in the heart of SHRSP. In conclusion, alterations in the expression of sphingolipid-metabolizing enzymes may be involved in the susceptibility to cardiac damage of hypertensive rat strains. Specific differences detected in the SHRSP, however, deserve further elucidation.

MST1 mediates doxorubicin-induced cardiomyopathy by SIRT3 downregulation.

Heart failure is a major side effect of doxorubicin (DOX) treatment in patients with cancer. However, the mechanisms underlying the development of DOX-induced heart failure need to be addressed. This study aims to test whether the serine/threonine kinase MST1, a major Hippo pathway component, contributes to the development of DOX-induced myocardial injury. C57BL/6J WT mice and mice with cardiomyocyte-specific dominant-negative MST1 (kinase-dead) overexpression received three weekly injections of DOX, reaching a final cumulative dose of 18 mg/kg. Echocardiographic, histological and biochemical analyses were performed six weeks after the first DOX administration. The effects of MST1 inhibition on DOX-induced cardiomyocyte injury were also tested in vitro. MST1 signaling was significantly activated in cardiomyocytes in response to DOX treatment in vitro and in vivo. Wild-type (WT) mice treated with DOX developed cardiac dysfunction and mitochondrial abnormalities. However, these detrimental effects were abolished in mice with cardiomyocyte-specific overexpression of dominant-negative MST1 (DN-MST1) or treated with XMU-MP-1, a specific MST1 inhibitor, indicating that MST1 inhibition attenuates DOX-induced cardiac dysfunction. DOX treatment led to a significant downregulation of cardiac levels of SIRT3, a deacetylase involved in mitochondrial protection, in WT mice, which was rescued by MST1 inhibition. Pharmacological inhibition of SIRT3 blunted the protective effects of MST1 inhibition, indicating that SIRT3 downregulation mediates the cytotoxic effects of MST1 activation in response to DOX treatment. Finally, we found a significant upregulation of MST1 and downregulation of SIRT3 levels in human myocardial tissue of cancer patients treated with DOX. In summary, MST1 contributes to DOX-induced cardiomyopathy through SIRT3 downregulation.

Effects of two-month treatment with a mixture of natural activators of autophagy on oxidative stress and arterial stiffness in patients with essential hypertension: A pilot study.

BACKGROUND AND AIMS: Trehalose, spermidine, nicotinamide, and polyphenols are natural substances that exert pro-autophagic and antioxidant properties. Their role in blood pressure (BP) regulation and preservation of vascular function in essential hypertension is unknown. The aim of this study was to evaluate the effect of a mixture of these agents on BP level, markers of oxidative stress, autophagy, endothelial function, and vascular stiffness in outpatients with grade 1 uncomplicated essential hypertension. METHODS AND RESULTS: A single-centre, open-label, case-control, pilot study was conducted in adult outpatients (aged ≥18 years) receiving or not the mixture for two months along with the standard therapies. Both at baseline and at the end of the treatment the following clinical parameters were evaluated: brachial seated office BP level, central aortic pressure, pulse wave velocity, augmentation index (AI@75). Both at baseline and at the end of the treatment, a blood sample was drawn for the measurement of: H2O2, HBA%, levels of sNOX2-dp, Atg 5, P62, endothelin 1, and NO bioavailability. The mixture of nutraceuticals did not influence BP levels. Patients receiving the mixture showed a significant decrease of oxidative stress, stimulation of autophagy, increased NO bioavailability and no increase of the AI@75, in contrast to what observed in hypertensive patients not receiving the mixture. CONCLUSIONS: The supplementation of the trehalose, spermidine, nicotinamide, and polyphenols mixture counteracted hypertension-related arterial stiffness through mechanisms likely dependent on oxidative stress downregulation and autophagy stimulation. These natural activators of autophagy may represent favourable adjuvants for prevention of the hypertensive cardiovascular damage.

Polymorphic variants at NDUFC2, encoding a mitochondrial complex I subunit, associate with cardiac hypertrophy in human hypertension.

BACKGROUND: A dysfunction of NADH dehydrogenase, the mitochondrial Complex I (CI), associated with the development of left ventricular hypertrophy (LVH) in previous experimental studies. A deficiency of Ndufc2 (subunit of CI) impairs CI activity causing severe mitochondrial dysfunction. The T allele at NDUFC2/rs11237379 variant associates with reduced gene expression and impaired mitochondrial function. The present study tested the association of both NDUFC2/rs11237379 and NDUFC2/rs641836 variants with LVH in hypertensive patients. In vitro studies explored the impact of reduced Ndufc2 expression in isolated cardiomyocytes. METHODS: Two-hundred-forty-six subjects (147 male, 59.7%), with a mean age of 59 ± 15 years, were included for the genetic association analysis. Ndufc2 silencing was performed in both H9c2 and rat primary cardiomyocytes to explore the hypertrophy development and the underlying signaling pathway. RESULTS: The TT genotype at NDUFC2/rs11237379 associated with significantly reduced gene expression. Multivariate analysis revealed that patients carrying this genotype showed significant differences for septal thickness (p = 0.07), posterior wall thickness (p = 0.008), RWT (p = 0.021), LV mass/BSA (p = 0.03), compared to subjects carrying either CC or CT genotypes. Patients carrying the A allele at NDUFC2/rs641836 showed significant differences for septal thickness (p = 0.017), posterior wall thickness (p = 0.011), LV mass (p = 0.003), LV mass/BSA (p = 0.002) and LV mass/height2.7(p = 0.010) after adjustment for covariates. In-vitro, the Ndufc2 deficiency-dependent mitochondrial dysfunction caused cardiomyocyte hypertrophy, pointing to SIRT3-AMPK-AKT-MnSOD as a major underlying signaling pathway. CONCLUSIONS: We demonstrated for the first time a significant association of NDUFC2 variants with LVH in human hypertension and highlight a key role of Ndufc2 deficiency-dependent CI mitochondrial dysfunction on increased susceptibility to cardiac hypertrophy development.

Virtual and Reality: A Neurophysiological Pilot Study of the Sarcophagus of the Spouses.

Art experience is not solely the observation of artistic objects, but great relevance is also placed on the environment in which the art experience takes place, often in museums and galleries. Interestingly, in the last few years, the introduction of some forms of virtual reality (VR) in museum contexts has been increasing. This has solicited enormous research interest in investigating any eventual differences between looking at the same artifact either in a real context (e.g. a museum) and in VR. To address such a target, a neuroaesthetic study was performed in which electroencephalography (EEG) and autonomic signals (heart rate and skin conductance) were recorded during the observation of the Etruscan artifact "Sarcophagus of the Spouses", both in the museum and in a VR reproduction. Results from EEG analysis showed a higher level of the Workload Index during observation in the museum compared to VR (p = 0.04), while the Approach-Withdrawal Index highlighted increased levels during the observation in VR compared to the observation in the museum (p = 0.03). Concerning autonomic indices, the museum elicited a higher Emotional Index response than the VR (p = 0.03). Overall, preliminary results suggest a higher engagement potential of the museum compared to VR, although VR could also favour higher embodiment than the museum.

Atrial natriuretic peptide stimulates autophagy/mitophagy and improves mitochondrial function in chronic heart failure.

Mitochondrial dysfunction, causing increased reactive oxygen species (ROS) production, is a molecular feature of heart failure (HF). A defective antioxidant response and mitophagic flux were reported in circulating leucocytes of patients with chronic HF and reduced ejection fraction (HFrEF). Atrial natriuretic peptide (ANP) exerts many cardiac beneficial effects, including the ability to protect cardiomyocytes by promoting autophagy. We tested the impact of ANP on autophagy/mitophagy, altered mitochondrial structure and function and increased oxidative stress in HFrEF patients by both ex vivo and in vivo approaches. The ex vivo study included thirteen HFrEF patients whose peripheral blood mononuclear cells (PBMCs) were isolated and treated with αANP (10-11 M) for 4 h. The in vivo study included six HFrEF patients who received sacubitril/valsartan for two months. PBMCs were characterized before and after treatment. Both approaches analyzed mitochondrial structure and functionality. We found that levels of αANP increased upon sacubitril/valsartan, whereas levels of NT-proBNP decreased. Both the ex vivo direct exposure to αANP and the higher αANP level upon in vivo treatment with sacubitril/valsartan caused: (i) improvement of mitochondrial membrane potential; (ii) stimulation of the autophagic process; (iii) significant reduction of mitochondrial mass-index of mitophagy stimulation-and upregulation of mitophagy-related genes; (iv) reduction of mitochondrial damage with increased inner mitochondrial membrane (IMM)/outer mitochondrial membrane (OMM) index and reduced ROS generation. Herein we demonstrate that αANP stimulates both autophagy and mitophagy responses, counteracts mitochondrial dysfunction, and damages ultimately reducing mitochondrial oxidative stress generation in PBMCs from chronic HF patients. These properties were confirmed upon sacubitril/valsartan administration, a pivotal drug in HFrEF treatment.

Beneficial Effects of Citrus Bergamia Polyphenolic Fraction on Saline Load-Induced Injury in Primary Cerebral Endothelial Cells from the Stroke-Prone Spontaneously Hypertensive Rat Model.

High salt load is a known noxious stimulus for vascular cells and a risk factor for cardiovascular diseases in both animal models and humans. The stroke-prone spontaneously hypertensive rat (SHRSP) accelerates stroke predisposition upon high-salt dietary feeding. We previously demonstrated that high salt load causes severe injury in primary cerebral endothelial cells isolated from SHRSP. This cellular model offers a unique opportunity to test the impact of substances toward the mechanisms underlying high-salt-induced vascular damage. We tested the effects of a bergamot polyphenolic fraction (BPF) on high-salt-induced injury in SHRSP cerebral endothelial cells. Cells were exposed to 20 mM NaCl for 72 h either in the absence or the presence of BPF. As a result, we confirmed that high salt load increased cellular ROS level, reduced viability, impaired angiogenesis, and caused mitochondrial dysfunction with a significant increase in mitochondrial oxidative stress. The addition of BPF reduced oxidative stress, rescued cell viability and angiogenesis, and recovered mitochondrial function with a significant decrease in mitochondrial oxidative stress. In conclusion, BPF counteracts the key molecular mechanisms underlying high-salt-induced endothelial cell damage. This natural antioxidant substance may represent a valuable adjuvant to treat vascular disorders.

Molecular mechanisms underlying the beneficial effects of exercise and dietary interventions in the prevention of cardiometabolic diseases.

Cardiometabolic diseases still represent a major cause of mortality worldwide. In addition to pharmacological approaches, lifestyle interventions can also be adopted for the prevention of these morbid conditions. Lifestyle changes include exercise and dietary restriction protocols, such as calorie restriction and intermittent fasting, which were shown to delay cardiovascular ageing and elicit health-promoting effects in preclinical models of cardiometabolic diseases. Beneficial effects are mediated by the restoration of multiple molecular mechanisms in heart and vessels that are compromised by metabolic stress. Exercise and dietary restriction rescue mitochondrial dysfunction, oxidative stress and inflammation. They also improve autophagy. The result of these effects is a marked improvement of vascular and heart function. In this review, we provide a comprehensive overview of the molecular mechanisms involved in the beneficial effects of exercise and dietary restriction in models of diabetes and obesity. We also discuss clinical studies and gap in animal-to-human translation.

1,5-disubstituted-1,2,3-triazoles counteract mitochondrial dysfunction acting on F1FO-ATPase in models of cardiovascular diseases.

The compromised viability and function of cardiovascular cells are rescued by small molecules of triazole derivatives (Tzs), identified as 3a and 3b, by preventing mitochondrial dysfunction. The oxidative phosphorylation improves the respiratory control rate in the presence of Tzs independently of the substrates that energize the mitochondria. The F1FO-ATPase, the main candidate in mitochondrial permeability transition pore (mPTP) formation, is the biological target of Tzs and hydrophilic F1 domain of the enzyme is depicted as the binding region of Tzs. The protective effect of Tz molecules on isolated mitochondria was corroborated by immortalized cardiomyocytes results. Indeed, mPTP opening was attenuated in response to ionomycin. Consequently, increased mitochondrial roundness and reduction of both length and interconnections between mitochondria. In in-vitro and ex-vivo models of cardiovascular pathologies (i.e., hypoxia-reoxygenation and hypertension) were used to evaluate the Tzs cardioprotective action. Key parameters of porcine aortic endothelial cells (pAECs) oxidative metabolism and cell viability were not affected by Tzs. However, in the presence of either 1 µM 3a or 0.5 µM 3b the impaired cell metabolism of pAECs injured by hypoxia-reoxygenation was restored to control respiratory profile. Moreover, endothelial cells isolated from SHRSP exposed to high-salt treatment rescued the Complex I activity and the endothelial capability to form vessel-like tubes and vascular function in presence of Tzs. As a result, the specific biochemical mechanism of Tzs to block Ca2+-activated F1FO-ATPase protected cell viability and preserved the pAECs bioenergetic metabolism upon hypoxia-reoxygenation injury. Moreover, SHRSP improved vascular dysfunction in response to a high-salt treatment.

NPPA/atrial natriuretic peptide is an extracellular modulator of autophagy in the heart.

NPPA/atrial natriuretic peptide (natriuretic peptide type A) exerts critical pleiotropic effects in the cardiovascular system, limiting cardiomyocyte hypertrophy and death, reducing cardiac fibrosis and promoting vascular integrity. However, the molecular mechanisms underlying these beneficial effects still need to be clarified. We demonstrated for the first time that macroautophagy/autophagy is involved in the local protective effects of NPPA in cardiomyocytes (CMs), both in vitro and in vivo. Exogenous NPPA rapidly activates autophagy in CMs through NPR1/type A natriuretic peptide receptor and PRKG/protein kinase G signaling and also increases cardiac autophagy in mice. Remarkably, endogenous NPPA is secreted by CMs in response to glucose deprivation or hypoxia, thereby stimulating autophagy through autocrine/paracrine mechanisms. NPPA preserves cell viability and reduces hypertrophy in response to stress through autophagy activation. In vivo, we found that Nppa knockout mice undergoing ischemia-reperfusion (I/R) show increased infarct size and reduced autophagy. Reactivation of autophagy by Tat-Beclin D11 limits I/R injury. We also found that the protective effects of NPPA in reducing infarct size are abrogated in the presence of autophagy inhibition. Mechanistically, we found that NPPA stimulates autophagy through the activation of TFEB (transcription factor EB). Our data suggest that NPPA is a novel extracellular regulator of autophagy in the heart.

Natural Activators of Autophagy Increase Maximal Walking Distance and Reduce Oxidative Stress in Patients with Peripheral Artery Disease: A Pilot Study.

Trehalose, spermidine, nicotinamide, and polyphenols have been shown to display pro-autophagic and antioxidant properties, eventually reducing cardiovascular and ischemic complications. This study aimed to investigate whether a mixture of these components improves maximal walking distance (MWD) in peripheral artery disease (PAD) patients. Nitrite/nitrate (NOx), endothelin-1, sNOX2-dp, H2O2 production, H2O2 break-down activity (HBA), ATG5 and P62 levels, flow-mediated dilation (FMD), and MWD were evaluated in 20 PAD patients randomly allocated to 10.5 g of mixture or no-treatment in a single-blind study. The above variables were assessed at baseline and 60 days after mixture ingestion. Compared with baseline, mixture intake significantly increased MWD (+91%; p < 0.01) and serum NOx (+96%; p < 0.001), whereas it significantly reduced endothelin-1 levels (−30%, p < 0.01). Moreover, mixture intake led to a remarkable reduction in sNOX2dp (−31%, p < 0.05) and H2O2 (−40%, p < 0.001) and potentiated antioxidant power (+110%, p < 0.001). Finally, mixture ingestion restored autophagy by increasing ATG5 (+43%, p < 0.01) and decreasing P62 (−29%, p < 0.05). No changes in the above-mentioned variables were observed in the no-treatment group. The treatment with a mixture of trehalose, spermidine, nicotinamide, and polyphenols improves MWD in PAD patients, with a mechanism possibly related to NOX2-mediated oxidative stress downregulation and autophagic flux upregulation. Clinical Trial Registration unique identifier: NCT04061070.

Mitochondria and Doxorubicin-Induced Cardiomyopathy: A Complex Interplay.

Cardiotoxicity has emerged as a major side effect of doxorubicin (DOX) treatment, affecting nearly 30% of patients within 5 years after chemotherapy. Heart failure is the first non-cancer cause of death in DOX-treated patients. Although many different molecular mechanisms explaining the cardiac derangements induced by DOX were identified in past decades, the translation to clinical practice has remained elusive to date. This review examines the current understanding of DOX-induced cardiomyopathy (DCM) with a focus on mitochondria, which were increasingly proven to be crucial determinants of DOX-induced cytotoxicity. We discuss DCM pathophysiology and epidemiology and DOX-induced detrimental effects on mitochondrial function, dynamics, biogenesis, and autophagy. Lastly, we review the current perspectives to contrast the development of DCM, which is still a relatively diffused, invalidating, and life-threatening condition for cancer survivors.

Role of Uncoupling Protein 2 Gene Polymorphisms on the Risk of Ischemic Stroke in a Sardinian Population.

The mitochondrial uncoupling protein 2 (UCP2) acts as an anion transporter and as an antioxidant factor able to reduce the reactive oxygen species level. Based on its effects, UCP2 prevents the membrane lipids, proteins, and DNA damage while preserving normal cellular functions. Many variants have been identified within the human UCP2. Some of them were associated with a higher risk of obesity, diabetes and cardiovascular diseases in different populations. UCP2 appears a suitable candidate also for the risk of ischemic stroke. In the current study, we investigated the possible association between few variants of UCP2 (rs659366, rs660339, rs1554995310) and the risk of ischemic stroke in a genetically homogenous cohort of cases and controls selected in Sardinia Island. This population has been previously analysed for other candidate genes. A total of 250 cases of ischemic stroke and 241 controls were enrolled in the study. The allelic/genotypic distribution of the 3 UCP2 variants was characterized and compared among cases and controls. The results of our study confirmed known risk factors for ischemic stroke: age, history of smoking, hypertension, hypercholesterolemia, and atrial fibrillation. No association was found between the 3 UCP2 variants and the risk of ischemic stroke in our Sardinian cohort.