Sebastiano Sciarretta

Both fusion and fission contribute to mitochondrial quality control. How unopposed fusion affects survival of cardiomyocytes and left ventricular function in the heart is poorly understood.We investigated the role of dynamin-related protein 1 (Drp1), a GTPase that mediates mitochondrial fission, in mediating mitochondrial autophagy, ventricular function, and stress resistance in the heart.Drp1 downregulation induced mitochondrial elongation, accumulation of damaged mitochondria, and increased apoptosis in cardiomyocytes at baseline. Drp1 downregulation also suppressed autophagosome formation and autophagic flux at baseline and in response to glucose deprivation in cardiomyocytes. The lack of lysosomal translocation of mitochondrially targeted Keima indicates that Drp1 downregulation suppressed mitochondrial autophagy. Mitochondrial elongation and accumulation of damaged mitochondria were also observed in tamoxifen-inducible cardiac-specific Drp1 knockout mice. After Drp1 downregulation, cardiac-specific Drp1 knockout mice developed left ventricular dysfunction, preceded by mitochondrial dysfunction, and died within 13 weeks. Autophagic flux is significantly suppressed in cardiac-specific Drp1 knockout mice. Although left ventricular function in cardiac-specific Drp1 heterozygous knockout mice was normal at 12 weeks of age, left ventricular function decreased more severely after 48 hours of fasting, and the infarct size/area at risk after ischemia/reperfusion was significantly greater in cardiac-specific Drp1 heterozygous knockout than in control mice.Disruption of Drp1 induces mitochondrial elongation, inhibits mitochondrial autophagy, and causes mitochondrial dysfunction, thereby promoting cardiac dysfunction and increased susceptibility to ischemia/reperfusion.

Here we show that Mst1, a proapoptotic kinase, impairs protein quality control mechanisms in the heart through inhibition of autophagy. Stress-induced activation of Mst1 in cardiomyocytes promoted accumulation of p62 and aggresome formation, accompanied by the disappearance of autophagosomes. Mst1 phosphorylated the Thr108 residue in the BH3 domain of Beclin1, which enhanced the interaction between Beclin1 and Bcl-2 and/or Bcl-xL, stabilized the Beclin1 homodimer, inhibited the phosphatidylinositide 3-kinase activity of the Atg14L-Beclin1-Vps34 complex and suppressed autophagy. Furthermore, Mst1-induced sequestration of Bcl-2 and Bcl-xL by Beclin1 allows Bax to become active, thereby stimulating apoptosis. Mst1 promoted cardiac dysfunction in mice subjected to myocardial infarction by inhibiting autophagy, associated with increased levels of Thr108-phosphorylated Beclin1. Moreover, dilated cardiomyopathy in humans was associated with increased levels of Thr108-phosphorylated Beclin1 and signs of autophagic suppression. These results suggest that Mst1 coordinately regulates autophagy and apoptosis by phosphorylating Beclin1 and consequently modulating a three-way interaction among Bcl-2 proteins, Beclin1 and Bax.

The protein kinase mammalian or mechanistic target of rapamycin (mTOR) is an atypical serine/threonine kinase that exerts its main cellular functions by interacting with specific adaptor proteins to form 2 different multiprotein complexes, mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2). mTORC1 regulates protein synthesis, cell growth and proliferation, autophagy, cell metabolism, and stress responses, whereas mTORC2 seems to regulate cell survival and polarity. The mTOR pathway plays a key regulatory function in cardiovascular physiology and pathology. However, the majority of information available about mTOR function in the cardiovascular system is related to the role of mTORC1 in the unstressed and stressed heart. mTORC1 is required for embryonic cardiovascular development and for postnatal maintenance of cardiac structure and function. In addition, mTORC1 is necessary for cardiac adaptation to pressure overload and development of compensatory hypertrophy. However, partial and selective pharmacological and genetic inhibition of mTORC1 was shown to extend life span in mammals, reduce pathological hypertrophy and heart failure caused by increased load or genetic cardiomyopathies, reduce myocardial damage after acute and chronic myocardial infarction, and reduce cardiac derangements caused by metabolic disorders. The optimal therapeutic strategy to target mTORC1 and increase cardioprotection is under intense investigation. This article reviews the information available regarding the effects exerted by mTOR signaling in cardiovascular physiology and pathological states.

Autophagy is an evolutionarily conserved mechanism by which cytoplasmic elements are degraded intracellularly. Autophagy has also emerged as a major regulator of cardiac homeostasis and function. Autophagy preserves cardiac structure and function under baseline conditions and is activated during stress, limiting damage under most conditions. It reduces injury and preserves cardiac function during ischemia. It also reduces chronic ischemic remodeling and mediates the cardiac adaptation to pressure overload by restricting misfolded protein accumulation, mitochondrial dysfunction, and oxidative stress. Impairment of autophagy is involved in the development of diabetes and aging-induced cardiac abnormalities. Autophagy defects contribute to the development of cardiac proteinopathy and doxorubicin-induced cardiomyopathy. However, massive activation of autophagy may be detrimental for the heart in certain stress conditions, such as reperfusion injury. In this review, we discuss recent evidence supporting the important role of autophagy and mitophagy in the regulation of cardiac homeostasis and adaptation to stress.

The mTOR (mechanistic target of rapamycin) is a master regulator of several crucial cellular processes, including protein synthesis, cellular growth, proliferation, autophagy, lysosomal function, and cell metabolism. mTOR interacts with specific adaptor proteins to form 2 multiprotein complexes, called mTORC1 (mTOR complex 1) and mTORC2 (mTOR complex 2). In the cardiovascular system, the mTOR pathway regulates both physiological and pathological processes in the heart. It is needed for embryonic cardiovascular development and for maintaining cardiac homeostasis in postnatal life. Studies involving mTOR loss-of-function models revealed that mTORC1 activation is indispensable for the development of adaptive cardiac hypertrophy in response to mechanical overload. mTORC2 is also required for normal cardiac physiology and ensures cardiomyocyte survival in response to pressure overload. However, partial genetic or pharmacological inhibition of mTORC1 reduces cardiac remodeling and heart failure in response to pressure overload and chronic myocardial infarction. In addition, mTORC1 blockade reduces cardiac derangements induced by genetic and metabolic disorders and has been reported to extend life span in mice. These studies suggest that pharmacological targeting of mTOR may represent a therapeutic strategy to confer cardioprotection, although clinical evidence in support of this notion is still scarce. This review summarizes and discusses the new evidence on the pathophysiological role of mTOR signaling in the cardiovascular system.

The aging population is increasing in developed countries. Because the incidence of cardiac disease increases dramatically with age, it is important to understand the molecular mechanisms through which the heart becomes either more or less susceptible to stress. Cardiac aging is characterized by the presence of hypertrophy, fibrosis, and accumulation of misfolded proteins and dysfunctional mitochondria. Macroautophagy (hereafter referred to as autophagy) is a lysosome-dependent bulk degradation mechanism that is essential for intracellular protein and organelle quality control. Autophagy and autophagic flux are generally decreased in aging hearts, and murine autophagy loss-of-function models develop exacerbated cardiac dysfunction that is accompanied by the accumulation of misfolded proteins and dysfunctional organelles. On the contrary, stimulation of autophagy generally improves cardiac function in mouse models of protein aggregation by removing accumulated misfolded proteins, dysfunctional mitochondria, and damaged DNA, thereby improving the overall cellular environment and alleviating aging-associated pathology in the heart. Increasing lines of evidence suggest that autophagy is required for many mechanisms that mediate lifespan extension, such as caloric restriction, in various organisms. These results raise the exciting possibility that autophagy may play an important role in combating the adverse effects of aging in the heart. In this review, we discuss the role of autophagy in the heart during aging, how autophagy alleviates age-dependent changes in the heart, and how the level of autophagy in the aging heart can be restored.

The vascular safety of electronic cigarettes (e-Cigarettes) must still be clarified. We compared the impact of e-Cigarettes vs traditional tobacco cigarettes on oxidative stress and endothelial function in healthy smokers and nonsmoker adults.A crossover, single-blind study was performed in 40 healthy subjects (20 smokers and 20 nonsmokers, matched for age and sex). First, all subjects smoked traditional tobacco cigarettes. One week later, the same subjects smoked an e-Cigarette with the same nominal nicotine content. Blood samples were drawn just before and after smoking, and markers of oxidative stress, nitric oxide bioavailability, and vitamin E levels were measured. Flow-mediated dilation (FMD) was also measured.Smoking both e-Cigarettes and traditional cigarettes led to a significant increase in the levels of soluble NOX2-derived peptide and 8-iso-prostaglandin F2α and a significant decrease in nitric oxide bioavailability, vitamin E levels, and FMD. Generalized estimating equation analysis confirmed that all markers of oxidative stress and FMD were significantly affected by smoking and showed that the biologic effects of e-Cigarettes vstraditional cigarettes on vitamin E levels (P = .413) and FMD (P = .311) were not statistically different. However, e-Cigarettes seemed to have a lesser impact than traditional cigarettes on levels of soluble NOX2-derived peptide (P = .001), 8-iso-prostaglandin F2α (P = .046), and nitric oxide bioavailability (P = .001).Our study showed that both cigarettes have unfavorable effects on markers of oxidative stress and FMD after single use, although e-Cigarettes seemed to have a lesser impact. Future studies are warranted to clarify the chronic vascular effects of e-Cigarette smoking.

Pathological molecular mechanisms involved in myocardial remodeling contribute to alter the existing structure of the heart, leading to cardiac dysfunction. Among the complex signaling network that characterizes myocardial remodeling, the distinct processes are myocyte loss, cardiac hypertrophy, alteration of extracellular matrix homeostasis, fibrosis, defective autophagy, metabolic abnormalities, and mitochondrial dysfunction. Several pathophysiological stimuli, such as pressure and volume overload, trigger the remodeling cascade, a process that initially confers protection to the heart as a compensatory mechanism. Yet chronic inflammation after myocardial infarction also leads to cardiac remodeling that, when prolonged, leads to heart failure progression. Here, we review the molecular pathways involved in cardiac remodeling, with particular emphasis on those associated with myocardial infarction. A better understanding of cell signaling involved in cardiac remodeling may support the development of new therapeutic strategies towards the treatment of heart failure and reduction of cardiac complications. We will also discuss data derived from gene therapy approaches for modulating key mediators of cardiac remodeling.

Rheb is a GTP-binding protein that promotes cell survival and mediates the cellular response to energy deprivation (ED). The role of Rheb in the regulation of cell survival during ED has not been investigated in the heart.Rheb is inactivated during cardiomyocyte (CM) glucose deprivation (GD) in vitro, and during acute myocardial ischemia in vivo. Rheb inhibition causes mTORC1 inhibition, because forced activation of Rheb, through Rheb overexpression in vitro and through inducible cardiac-specific Rheb overexpression in vivo, restored mTORC1 activity. Restoration of mTORC1 activity reduced CM survival during GD and increased infarct size after ischemia, both of which were accompanied by inhibition of autophagy, whereas Rheb knockdown increased autophagy and CM survival. Rheb inhibits autophagy mostly through Atg7 depletion. Restoration of autophagy, through Atg7 reexpression and inhibition of mTORC1, increased cellular ATP content and reduced endoplasmic reticulum stress, thereby reducing CM death induced by Rheb activation. Mice with high-fat diet-induced obesity and metabolic syndrome (HFD mice) exhibited deregulated cardiac activation of Rheb and mTORC1, particularly during ischemia. HFD mice presented inhibition of cardiac autophagy and displayed increased ischemic injury. Pharmacological and genetic inhibition of mTORC1 restored autophagy and abrogated the increase in infarct size observed in HFD mice, but they failed to protect HFD mice in the presence of genetic disruption of autophagy.Inactivation of Rheb protects CMs during ED through activation of autophagy. Rheb and mTORC1 may represent therapeutic targets to reduce myocardial damage during ischemia, particularly in obese patients.

The Hippo pathway is an evolutionarily conserved regulator of organ size and tumorigenesis that negatively regulates cell growth and survival. Here we report that Yes-associated protein (YAP), the terminal effector of the Hippo pathway, interacts with FoxO1 in the nucleus of cardiomyocytes, thereby promoting survival. YAP and FoxO1 form a functional complex on the promoters of the catalase and manganese superoxide dismutase (MnSOD) antioxidant genes and stimulate their transcription. Inactivation of YAP, induced by Hippo activation, suppresses FoxO1 activity and decreases antioxidant gene expression, suggesting that Hippo signalling modulates the FoxO1-mediated antioxidant response. In the setting of ischaemia/reperfusion (I/R) in the heart, activation of Hippo antagonizes YAP-FoxO1, leading to enhanced oxidative stress-induced cell death through downregulation of catalase and MnSOD. Conversely, restoration of YAP activity protects against I/R injury. These results suggest that YAP is a nuclear co-factor of FoxO1 and that the Hippo pathway negatively affects cardiomyocyte survival by inhibiting the function of YAP-FoxO1. YAP is the terminal effector of the Hippo signalling pathway that regulates cell growth and survival. Shao et al.show that Hippo signalling also stimulates oxidative stress responses in cardiomyocytes, revealing YAP as a regulator of FoxO1-dependent antioxidant gene expression.

The aim of this study was to examine the long-term prognosis in asymptomatic individuals with a coronary artery calcium (CAC) score of 0 and its associated warranty period.Emerging evidence supports a CAC score of 0 as a favorable cardiovascular short-to intermediate-term prognostic factor.A total of 9,715 individuals undergoing CAC imaging were stratified by age, Framingham risk score (FRS), and National Cholesterol Education Program Adult Treatment Panel III (NCEP ATP III) categories and followed for a mean of 14.6 years (range 12.9 to 16.8 years). Cox regression, area under the receiver-operating characteristic curve, and net reclassification information were used to assess all-cause mortality, discrimination, and reclassification of a CAC score of 0 compared with the FRS and NCEP ATP III, respectively. A warranty period was pre-defined as <1% annual mortality rate. Vascular age was estimated by linear regression.In 4,864 individuals with a baseline CAC score of 0 (mean age, 52.1 ± 10.8 years; 57.9% male), 229 deaths occurred. The warranty period of a CAC score of 0 was almost 15 years for individuals at low and intermediate risk with no significant differences regarding age and sex. A CAC score of 0 was associated with a vascular age of 1, 10, 20, and 30 years less than the chronological age of individuals between 50 and 59, 60 and 69, 70 and 79, and 80 years of age and older, respectively. The CAC score was the strongest predictor of death (hazard ratio: 2.67, 95% confidence interval: 2.29 to 3.11) that enabled discrimination and consistent reclassification beyond the FRS (area under the receiver-operating characteristic curve: 0.71 vs. 0.64, p < 0.001) and NCEP ATP III (area under the receiver-operating characteristic curve: 0.72 vs. 0.64, p < 0.001).A CAC score of 0 confers a 15-year warranty period against mortality in individuals at low to intermediate risk that is unaffected by age or sex. Furthermore, in individuals considered at high risk by clinical risk scores, a CAC score of 0 confers better survival than in individuals at low to intermediate risk but with any CAC score.

5'-AMP-activated protein kinase (AMPK) is a key regulator of metabolism and survival during energy stress. Dysregulation of AMPK is strongly associated with oxidative-stress-related disease. However, whether and how AMPK is regulated by intracellular redox status remains unknown. Here we show that the activity of AMPK is negatively regulated by oxidation of Cys130 and Cys174 in its α subunit, which interferes with the interaction between AMPK and AMPK kinases (AMPKK). Reduction of Cys130/Cys174 is essential for activation of AMPK during energy starvation. Thioredoxin1 (Trx1), an important reducing enzyme that cleaves disulfides in proteins, prevents AMPK oxidation, serving as an essential cofactor for AMPK activation. High-fat diet consumption downregulates Trx1 and induces AMPK oxidation, which enhances cardiomyocyte death during myocardial ischemia. Thus, Trx1 modulates activation of the cardioprotective AMPK pathway during ischemia, functionally linking oxidative stress and metabolism in the heart.

Trehalose (TRE) is a natural, nonreducing disaccharide synthesized by lower organisms. TRE exhibits an extraordinary ability to protect cells against different kinds of stresses through activation of autophagy. However, the effect of TRE on the heart during stress has never been tested. This study evaluated the effects of TRE administration in a mouse model of chronic ischemic remodeling. Wild-type (WT) or beclin 1+/− mice were subjected to permanent ligation of the left anterior descending artery (LAD) and then treated with either placebo or trehalose (1 mg/g/day intraperitoneally for 48 h, then 2% in the drinking water). After 4 weeks, echocardiographic, hemodynamic, gravimetric, histological, and biochemical analyses were conducted. TRE reduced left ventricular (LV) dilation and increased ventricular function in mice with LAD ligation compared with placebo. Sucrose, another nonreducing disaccharide, did not exert protective effects during post-infarction LV remodeling. Trehalose administration to mice overexpressing GFP-tagged LC3 significantly increased the number of GFP-LC3 dots, both in the presence and absence of chloroquine administration. TRE also increased cardiac LC3-II levels after 4 weeks following myocardial infarction (MI), indicating that it induced autophagy in the heart in vivo. To evaluate whether TRE exerted beneficial effects through activation of autophagy, trehalose was administered to beclin 1+/− mice. The improvement of LV function, lung congestion, cardiac remodeling, apoptosis, and fibrosis following TRE treatment observed in WT mice were all significantly blunted in beclin 1+/− mice. TRE reduced MI-induced cardiac remodeling and dysfunction through activation of autophagy.

Energy stress, such as ischemia, induces mitochondrial damage and death in the heart. Degradation of damaged mitochondria by mitophagy is essential for the maintenance of healthy mitochondria and survival. Here, we show that mitophagy during myocardial ischemia was mediated predominantly through autophagy characterized by Rab9-associated autophagosomes, rather than the well-characterized form of autophagy that is dependent on the autophagy-related 7 (Atg) conjugation system and LC3. This form of mitophagy played an essential role in protecting the heart against ischemia and was mediated by a protein complex consisting of unc-51 like kinase 1 (Ulk1), Rab9, receptor-interacting serine/thronine protein kinase 1 (Rip1), and dynamin-related protein 1 (Drp1). This complex allowed the recruitment of trans-Golgi membranes associated with Rab9 to damaged mitochondria through S179 phosphorylation of Rab9 by Ulk1 and S616 phosphorylation of Drp1 by Rip1. Knockin of Rab9 (S179A) abolished mitophagy and exacerbated the injury in response to myocardial ischemia, without affecting conventional autophagy. Mitophagy mediated through the Ulk1/Rab9/Rip1/Drp1 pathway protected the heart against ischemia by maintaining healthy mitochondria.

Heart failure is a highly morbid and mortal clinical condition that represents the last stage of most cardiovascular disorders. Diabetes is strongly associated with an increased incidence of heart failure and directly promotes cardiac hypertrophy, fibrosis, and apoptosis. These changes, in turn, contribute to the development of ventricular dysfunction. The clinical condition associated with the spectrum of cardiac abnormalities induced by diabetes is termed diabetic cardiomyopathy. Myocardial inflammation has recently emerged as a pathophysiological process contributing to cardiac hypertrophy, fibrosis, and dysfunction in cardiac diseases. Myocardial inflammation is also implicated in the development of diabetic cardiomyopathy. Several molecular mechanisms link diabetes to myocardial inflammation. The NF-κB signalling pathway and the renin-angiotensin-aldosterone system are strongly activated in the diabetic heart, thereby promoting myocardial inflammation. Advanced glycation end-products and damage-associated molecular pattern molecules also represent strong triggers for inflammation. The mediators resulting from this inflammatory process modulate specific intracellular signalling mechanisms in cardiac cells that promote the development of diabetic cardiomyopathy. This review article will provide an overview of the signalling molecular mechanisms linking diabetic cardiomyopathy to myocardial inflammation.

Background: Anthracyclines, such as doxorubicin (DOX), are potent anticancer agents for the treatment of solid tumors and hematologic malignancies. However, their clinical use is hampered by cardiotoxicity. This study sought to investigate the role of phosphoinositide 3-kinase γ (PI3Kγ) in DOX-induced cardiotoxicity and the potential cardioprotective and anticancer effects of PI3Kγ inhibition. Methods: Mice expressing a kinase-inactive PI3Kγ or receiving PI3Kγ-selective inhibitors were subjected to chronic DOX treatment. Cardiac function was analyzed by echocardiography, and DOX-mediated signaling was assessed in whole hearts or isolated cardiomyocytes. The dual cardioprotective and antitumor action of PI3Kγ inhibition was assessed in mouse mammary tumor models. Results: PI3Kγ kinase-dead mice showed preserved cardiac function after chronic low-dose DOX treatment and were protected against DOX-induced cardiotoxicity. The beneficial effects of PI3Kγ inhibition were causally linked to enhanced autophagic disposal of DOX-damaged mitochondria. Consistently, either pharmacological or genetic blockade of autophagy in vivo abrogated the resistance of PI3Kγ kinase-dead mice to DOX cardiotoxicity. Mechanistically, PI3Kγ was triggered in DOX-treated hearts, downstream of Toll-like receptor 9, by the mitochondrial DNA released by injured organelles and contained in autolysosomes. This autolysosomal PI3Kγ/Akt/mTOR/Ulk1 signaling provided maladaptive feedback inhibition of autophagy. PI3Kγ blockade in models of mammary gland tumors prevented DOX-induced cardiac dysfunction and concomitantly synergized with the antitumor action of DOX by unleashing anticancer immunity. Conclusions: Blockade of PI3Kγ may provide a dual therapeutic advantage in cancer therapy by simultaneously preventing anthracyclines cardiotoxicity and reducing tumor growth.

The process of mitochondrial dynamics is emerging as a core player in cardiovascular homeostasis. This process refers to the co‐ordinated cycles of biogenesis, fusion, fission and degradation to which mitochondria constantly undergo to maintain their integrity, distribution and size. These mechanisms represent an early response to mitochondrial stress, confining organelle portions that are irreversibly damaged and preserving mitochondrial function. Accumulating evidence demonstrates that impairment in mitochondrial dynamics leads to myocardial damage and cardiac disease progression in a variety of disease models, including pressure overload, ischaemia/reperfusion and metabolic disturbance. These findings suggest that modulation of mitochondrial dynamics may be considered as a valid therapeutic strategy in cardiovascular diseases. In this review, we discuss the current evidence about the role of mitochondrial dynamics in cardiac pathophysiology, with a particular focus on the mechanisms underlying the development of cardiac hypertrophy and heart failure, metabolic and genetic cardiomyopathies, ischaemia/reperfusion injury, atherosclerosis and ischaemic stroke. LINKED ARTICLES This article is part of a themed issue on Cellular metabolism and diseases. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v178.10/issuetoc

Background Little clinical research on new-generation heat-not-burn cigarettes ( HNBC ) in comparison with electronic vaping cigarettes ( EVC ) and traditional tobacco combustion cigarettes ( TC ) has been reported. We aimed to appraise the acute effects of single use of HNBC , EVC, and TC in healthy smokers. Methods and Results This was an independent, cross-over, randomized trial in 20 TC smokers, with allocation to different cycles of HNBC , EVC , and TC . All participants used all types of products, with an intercycle washout of 1 week. End points were oxidative stress, antioxidant reserve, platelet activation, flow-mediated dilation, blood pressure, and satisfaction scores. Single use of any product led to an adverse impact on oxidative stress, antioxidant reserve, platelet function, flow-mediated dilation, and blood pressure. HNBC had less impact than EVC and TC on soluble Nox2-derived peptide (respectively, P=0.004 and 0.001), 8-iso-prostaglandin F2α- III ( P=0.004 and <0.001), and vitamin E ( P=0.018 and 0.044). HNBC and EVC were equally less impactful than TCs on flow-mediated dilation ( P=0.872 for HNBC versus EVC ), H2O2 ( P=0.522), H2O2 breakdown activity ( P=0.091), soluble CD 40 ligand ( P=0.849), and soluble P-selectin ( P=0.821). The effect of HNBC and, to a lesser extent EVC , on blood pressure was less evident than that of TC , whereas HNBC appeared more satisfying than EVC (all P<0.05). Conclusions Acute effects of HNBC , EVC, and TC are different on several oxidative stress, antioxidant reserve, platelet function, cardiovascular, and satisfaction dimensions, with TCs showing the most detrimental changes in clinically relevant features. Clinical Trial Registration URL : http://www.clinicaltrials.gov . Unique identifier: NCT 03301129.

Rationale: Inhibition of glycogen synthase kinase-3 (GSK-3) protects the heart during ischemia/reperfusion (I/R), yet the underlying mechanisms of cardioprotection afforded by beta isoform-specific inhibition GSK-3 remain to be elucidated. Objective: We studied the molecular mechanism mediating the effect of GSK-3β activation/inhibition upon myocardial injury during prolonged ischemia and I/R. Methods and Results: Beta isoform–specific inhibition of GSK-3 by dominant negative GSK-3β in transgenic mice (Tg-DnGSK-3β) or in heterozygous GSK-3β knock-out mice (GSK-3β+/−) significantly increased, whereas activation of GSK-3β in constitutively active GSK-3β knock-in mice (βKI) significantly decreased, myocardial ischemic injury after prolonged ischemia. In contrast, inhibition of GSK-3β in Tg-DnGSK-3β or GSK-3β+/− significantly reduced, while activation of GSK-3β in βKI significantly enhanced, myocardial I/R injury. Inhibition of GSK-3β stimulated mTOR signaling and inhibited autophagy through a rapamycin-sensitive (mTOR dependent) mechanism. Rapamycin enhanced autophagy and, at the same time, abolished the effects of GSK-3β inhibition on both prolonged ischemic injury and I/R injury. Importantly, the influence of rapamycin over the effects of GSK-3β inhibition on myocardial injury was reversed by inhibition of autophagy. Conclusions: Our results suggest that beta isoform–specific inhibition of GSK-3 exacerbates ischemic injury but protects against I/R injury by modulating mTOR and autophagy.

Autophagy is an essential survival mechanism during energy stress in the heart. Oxidative stress is activated by energy stress, but its role in mediating autophagy is poorly understood. NADPH oxidase (Nox) 4 is an enzyme that generates reactive oxygen species (ROS) at intracellular membranes. Whether Nox4 acts as a sensor of energy stress to mediate activation of autophagy is unknown.We investigated whether Nox4 is involved in the regulation of autophagy and cell survival during energy stress in cardiomyocytes.Production of ROS in cardiomyocytes was increased during glucose deprivation (GD) in a Nox4-dependent manner. Protein levels and the ROS-producing activity of Nox4 were increased in the endoplasmic reticulum (ER), but not in mitochondria, in response to GD. Selective knockdown of Nox4, but not Nox2, or selective reduction of ROS in the ER with ER-targeted catalase, but not mitochondria-targeted perioxiredoxin 3, abrogated GD-induced autophagy. Nox4 promoted autophagy during GD through activation of the protein kinase RNA-activated-like ER kinase pathway by suppression of prolyl hydroxylase 4. The decrease in cell survival during GD in the presence of Nox4 knockdown was rescued by reactivation of autophagy by Atg7 overexpression, indicating that the effect of Nox4 on cell survival is critically mediated through regulation of autophagy. Nox4 was activated during fasting and prolonged ischemia in the mouse heart, where Nox4 is also required for autophagy activation and cardioprotection.Nox4 critically mediates autophagy in response to energy stress in cardiomyocytes by eliciting ROS in the ER and stimulating the protein kinase RNA-activated-like ER kinase signaling pathway.

Autophagy is a catabolic process that degrades long-lived proteins and damaged organelles by sequestering them into double membrane structures termed “autophagosomes” and fusing them with lysosomes. Autophagy is active in the heart at baseline and further stimulated under stress conditions including starvation, ischemia/reperfusion, and heart failure. It plays an adaptive role in the heart at baseline, thereby maintaining cardiac structure and function and inhibiting age-related cardiac abnormalities. Autophagy is activated by ischemia and nutrient starvation in the heart through Sirt1-FoxO- and adenosine monophosphate (AMP)-activated protein kinase (AMPK)-dependent mechanisms, respectively. Activation of autophagy during ischemia is essential for cell survival and maintenance of cardiac function. Autophagy is strongly activated in the heart during reperfusion after ischemia. Activation of autophagy during reperfusion could be either protective or detrimental, depending on the experimental model. However, strong induction of autophagy accompanied by robust upregulation of Beclin1 could cause autophagic cell death, thereby proving to be detrimental. This review provides an overview regarding both protective and detrimental functions of autophagy in the heart and discusses possible applications of current knowledge to the treatment of heart disease.

The Difficult Task of Finding the Best Antihypertensive Agent: Comment on “Antihypertensive Treatment and Development of Heart Failure in Hypertension”

Rationale: In Drosophila , the Hippo signaling pathway negatively regulates organ size by suppressing cell proliferation and survival through the inhibition of Yorkie, a transcriptional cofactor. Yes-associated protein (YAP), the mammalian homolog of Yorkie, promotes cardiomyocyte growth and survival in postnatal hearts. However, the underlying mechanism responsible for the beneficial effect of YAP in cardiomyocytes remains unclear. Objectives: We investigated whether miR-206, a microRNA known to promote hypertrophy in skeletal muscle, mediates the effect of YAP on promotion of survival and hypertrophy in cardiomyocytes. Methods and Results: Microarray analysis indicated that YAP increased miR-206 expression in cardiomyocytes. Increased miR-206 expression induced cardiac hypertrophy and inhibited cell death in cultured cardiomyocytes, similar to that of YAP. Downregulation of endogenous miR-206 in cardiomyocytes attenuated YAP-induced cardiac hypertrophy and survival, suggesting that miR-206 plays a critical role in mediating YAP function. Cardiac-specific overexpression of miR-206 in mice induced hypertrophy and protected the heart from ischemia/reperfusion injury, whereas suppression of miR-206 exacerbated ischemia/reperfusion injury and prevented pressure overload-induced cardiac hypertrophy. miR-206 negatively regulates Forkhead box protein P1 expression in cardiomyocytes and overexpression of Forkhead box protein P1 attenuated miR-206–induced cardiac hypertrophy and survival, suggesting that Forkhead box protein P1 is a functional target of miR-206. Conclusions: YAP increases the abundance of miR-206, which in turn plays an essential role in mediating hypertrophy and survival by silencing Forkhead box protein P1 in cardiomyocytes.

Left ventricular diastolic dysfunction represents a frequent clinical condition and is associated with increased cardiovascular morbidity and mortality. Diastolic dysfunction is the most common cause of HF-PSF (heart failure with preserved ejection fraction). Therefore it becomes important to understand the pathophysiological mechanisms underlying diastolic dysfunction, as well as the effective therapeutic strategies able to antagonize its development and progression. Among the complex pathophysiological factors that may contribute to the development of diastolic dysfunction, the RAAS (renin–angiotensin–aldosterone system) has been shown to play a significant role. Paracrine and autocrine signals of the RAAS promote structural and functional changes in the heart largely linked to increased myocardial fibrosis. Enhanced and dysregulated activity of the RAAS also contributes to the development of volume overload and vasoconstriction with subsequent increases in left ventricular diastolic filling pressures and a higher susceptibility of developing CHF (congestive heart failure). More recently, it has also been suggested that the RAAS may play a role in triggering myocardial and vascular inflammation through the activation of different cell types and the secretion of cytokines and chemokines. RAAS-induced myocardial inflammation leads to perivascular myocardial fibrosis and to the development or progression of diastolic dysfunction. For these reasons pharmacological blockade of the RAAS has been proposed as a rational approach for the treatment of diastolic dysfunction. In fact, ACEIs (angiotensin-converting enzyme inhibitors), ARBs (angiotensin II receptor blockers) and AAs (aldosterone antagonists) have been demonstrated to delay the development and progression from pre-clinical diastolic dysfunction towards CHF, as well as to reduce the morbidity and mortality associated with this condition.

The mTOR and Hippo pathways have recently emerged as the major signaling transduction cascades regulating organ size and cellular homeostasis. However, direct crosstalk between two pathways is yet to be determined. Here, we demonstrate that mTORC2 is a direct negative regulator of the MST1 kinase, a key component of the Hippo pathway. mTORC2 phosphorylates MST1 at serine 438 in the SARAH domain, thereby reducing its homodimerization and activity. We found that Rictor/mTORC2 preserves cardiac structure and function by restraining the activity of MST1 kinase. Cardiac-specific mTORC2 disruption through Rictor deletion leads to a marked activation of MST1 that, in turn, promotes cardiac dysfunction and dilation, impairing cardiac growth and adaptation in response to pressure overload. In conclusion, our study demonstrates the existence of a direct crosstalk between mTORC2 and MST1 that is critical for cardiac cell survival and growth.

Rationale: NF2 (neurofibromin 2) is an established tumor suppressor that promotes apoptosis and inhibits growth in a variety of cell types, yet its function in cardiomyocytes remains largely unknown. Objective: We sought to determine the role of NF2 in cardiomyocyte apoptosis and ischemia/reperfusion (I/R) injury in the heart. Methods and Results: We investigated the function of NF2 in isolated cardiomyocytes and mouse myocardium at baseline and in response to oxidative stress. NF2 was activated in cardiomyocytes subjected to H 2 O 2 and in murine hearts subjected to I/R. Increased NF2 expression promoted the activation of Mst1 (mammalian sterile 20–like kinase 1) and the inhibition of Yap (Yes-associated protein), whereas knockdown of NF2 attenuated these responses after oxidative stress. NF2 increased the apoptosis of cardiomyocytes that appeared dependent on Mst1 activity. Mice deficient for NF2 in cardiomyocytes, NF2 cardiomyocyte-specific knockout (CKO), were protected against global I/R ex vivo and showed improved cardiac functional recovery. Moreover, NF2 cardiomyocyte-specific knockout mice were protected against I/R injury in vivo and showed the upregulation of Yap target gene expression. Mechanistically, we observed nuclear association between NF2 and its activator MYPT-1 (myosin phosphatase target subunit 1) in cardiomyocytes, and a subpopulation of stress-induced nuclear Mst1 was diminished in NF2 CKO hearts. Finally, mice deficient for both NF2 and Yap failed to show protection against I/R indicating that Yap is an important target of NF2 in the adult heart. Conclusions: NF2 is activated by oxidative stress in cardiomyocytes and mouse myocardium and facilitates apoptosis. NF2 promotes I/R injury through the activation of Mst1 and inhibition of Yap, thereby regulating Hippo signaling in the adult heart.

The heart is highly sensitive to the aging process. In the elderly, the heart tends to become hypertrophic and fibrotic. Stiffness increases with ensuing systolic and diastolic dysfunction. Aging also affects the cardiac response to stress. At the molecular level, the aging process is associated with accumulation of damaged proteins and organelles, partially due to defects in protein quality control systems. The accumulation of dysfunctional and abnormal mitochondria is an important pathophysiological feature of the aging process, which is associated with excessive production of reactive oxygen species. Mitochondrial fusion and fission and mitochondrial autophagy are crucial mechanisms for maintaining mitochondrial function and preserving energy production. In particular, mitochondrial fission allows for selective segregation of damaged mitochondria, which are afterward eliminated by autophagy. Unfortunately, recent evidence indicates that mitochondrial dynamics and autophagy are progressively impaired over time, contributing to the aging process. This suggests that restoration of these mechanisms could delay organ senescence and prevent age-associated cardiac diseases. Here, we discuss the current understanding of the close relationship between mitochondrial dynamics, mitophagy, oxidative stress, and aging, with a particular focus on the heart.

Abstract Experimental studies showed that gut-derived lipopolysaccharide (LPS) is pro-atherogenic, however, its relationship with human atherosclerosis is still to be defined. We investigate if gut-derived LPS from Escherichia Coli localizes in human carotid plaque and its potential role as pro-inflammatory molecule in the atherosclerotic lesion. LPS from Escherichia Coli and Toll-like receptor 4 (TLR4) were studied in specimens from carotid and thyroid arteries of 10 patients undergoing endarterectomy and 15 controls matched for demographic and clinical characteristics. Blood LPS were significantly higher in patients compared to controls. Immunochemistry analysis revealed positivity for antibodies against LPS and TLR4 coincidentally with positivity for CD68 only in the atherosclerotic plaque of carotid arteries but not in thyroid arteries; the positivity for LPS and TLR4 was greater in the area with activated macrophages. LPS concentration similar to that detected in atherosclerotic plaque resulted in a dose-dependent TLR4-mediated Nox2 up-regulation by human monocytes. These data provide the first evidence that LPS from Escherichia Coli localizes in human plaque and may contribute to atherosclerotic damage via TLR4-mediated oxidative stress.

Rationale: The Hippo pathway plays an important role in determining organ size through regulation of cell proliferation and apoptosis. Hippo inactivation and consequent activation of YAP (Yes-associated protein), a transcription cofactor, have been proposed as a strategy to promote myocardial regeneration after myocardial infarction. However, the long-term effects of Hippo deficiency on cardiac function under stress remain unknown. Objective: We investigated the long-term effect of Hippo deficiency on cardiac function in the presence of pressure overload (PO). Methods and Results: We used mice with cardiac-specific homozygous knockout of WW45 (WW45cKO), in which activation of Mst1 (Mammalian sterile 20-like 1) and Lats2 (large tumor suppressor kinase 2), the upstream kinases of the Hippo pathway, is effectively suppressed because of the absence of the scaffolding protein. We used male mice at 3 to 4 month of age in all animal experiments. We subjected WW45cKO mice to transverse aortic constriction for up to 12 weeks. WW45cKO mice exhibited higher levels of nuclear YAP in cardiomyocytes during PO. Unexpectedly, the progression of cardiac dysfunction induced by PO was exacerbated in WW45cKO mice, despite decreased apoptosis and activated cardiomyocyte cell cycle reentry. WW45cKO mice exhibited cardiomyocyte sarcomere disarray and upregulation of TEAD1 (transcriptional enhancer factor) target genes involved in cardiomyocyte dedifferentiation during PO. Genetic and pharmacological inactivation of the YAP-TEAD1 pathway reduced the PO-induced cardiac dysfunction in WW45cKO mice and attenuated cardiomyocyte dedifferentiation. Furthermore, the YAP-TEAD1 pathway upregulated OSM (oncostatin M) and OSM receptors, which played an essential role in mediating cardiomyocyte dedifferentiation. OSM also upregulated YAP and TEAD1 and promoted cardiomyocyte dedifferentiation, indicating the existence of a positive feedback mechanism consisting of YAP, TEAD1, and OSM. Conclusions: Although activation of YAP promotes cardiomyocyte regeneration after cardiac injury, it induces cardiomyocyte dedifferentiation and heart failure in the long-term in the presence of PO through activation of the YAP-TEAD1-OSM positive feedback mechanism.

A growing number of evidences report that aging represents the major risk factor for the development of cardio and cerebrovascular diseases.Understanding Aging from a genetic, biochemical and physiological point of view could be helpful to design a better medical approach and to elaborate the best therapeutic strategy to adopt, without neglecting all the risk factors associated with advanced age.Of course, the better way should always be understanding risk-to-benefit ratio, maintenance of independence and reduction of symptoms.Although improvements in treatment of cardiovascular diseases in the elderly population have increased the survival rate, several studies are needed to understand the best management option to improve therapeutic outcomes.The aim of this review is to give a 360 • panorama on what goes on in the fragile ecosystem of elderly, why it happens and what we can do, right now, with the tools at our disposal to slow down aging, until new discoveries on aging, cardio and cerebrovascular diseases are at hand.

Abstract Aims Low-grade endotoxaemia is detectable in human circulation but its role in thrombosis is still unclear. Methods and results We measured serum lipopolysaccharide (LPS) concentration, soluble P-selectin (sP-selectin), a marker of platelet activation, and zonulin, a marker of gut permeability, in peripheral circulation, coronary thrombi, and intracoronary blood of patients with ST-elevation myocardial infarction (STEMI, n = 50) and stable angina (SA) (n = 50), respectively, and in controls (n = 50). Experimental study was carried out in mice to assess if Escherichia coli-LPS (E. coli-LPS) possess thrombotic property. Coronary thrombi from STEMI showed higher concentrations of LPS, sP-selectin vs. intracoronary blood of SA and peripheral blood of controls (P &amp;lt; 0.001). Zonulin was higher in STEMI compared to the other two groups [4.57 (3.34–5.22); 2.56 (0.41–4.36); 1.95 (1.22–2.65) ng/mL; P &amp;lt; 0.001] and correlated with LPS (Rs = 0.585; P &amp;lt; 0.001). Escherichia coli DNA was positive in 34% of STEMI vs. 12% of SA and 4% of controls (P &amp;lt; 0.001). In a subgroup of 12 STEMI, immunohistochemical analysis of coronary thrombi showed positivity for leucocyte Toll-like receptor 4 (TLR4), cathepsin G, and LPS from E. coli in 100%, 80%, and 25% of samples, respectively. E. coli-LPS injected in mice to reach LPS concentrations like those detected in coronary thrombi was associated with enhanced artery thrombosis and platelet activation, an effect blunted by TLR4 inhibitor co-administration. In vitro study demonstrated that LPS from E. coli enhanced platelet aggregation via TLR4-mediated leucocyte cathepsin G activation. Conclusion ST-elevation myocardial infarction patients disclose an enhanced gut permeability that results in LPS translocation in human circulation and eventually thrombus growth at site of artery lesion via leucocyte–platelet interaction.

Within the family of endogenous gasotransmitters, nitric oxide (NO) is the smallest gaseous intercellular messenger involved in the modulation of several processes, such as blood flow and platelet aggregation control, essential to maintain vascular homeostasis. NO is produced by nitric oxide synthases (NOS) and its effects are mediated by cGMP-dependent or cGMP-independent mechanisms. Growing evidence suggests a crosstalk between the NO signaling and the occurrence of oxidative stress in the onset and progression of vascular diseases, such as hypertension, heart failure, ischemia, and stroke. For these reasons, NO is considered as an emerging molecular target for developing therapeutic strategies for cardio- and cerebrovascular pathologies. Several natural derived compounds, such as polyphenols, are now proposed as modulators of NO-mediated pathways. The aim of this review is to highlight the experimental evidence on the involvement of nitric oxide in vascular homeostasis focusing on the therapeutic potential of targeting NO with some natural compounds in patients with vascular diseases.

Electronic cigarettes (E-cigarettes) have become popular as substitutes for conventional tobacco cigarettes or to aid quitting, but little is known about the potential risks to cardiovascular health for smokers and nonsmokers. We sought to compare the impact of E-cigarettes with conventional cigarettes on platelet function in healthy adult smokers and nonsmokers. A crossover single-blind study in 40 healthy participants (20 smokers, 20 nonsmokers, matched for age and sex) was conducted. Each participant smoked a conventional cigarette then returned 1 week later to vape a study E-cigarette with the same nominal nicotine content. Blood samples were drawn shortly before and 5 minutes after each episode and analyzed for platelet aggregation, soluble CD40-ligand (sCD40L) and soluble P-selectin (sP-selectin). At baseline, smokers had significantly higher levels of sCD40L and sP-selectin (all p ≤0.01) than nonsmokers. Within 5 minutes of using either a conventional cigarette or E-cigarette, changes in the levels of sCD40L, sP-selectin, and platelet aggregation (all p ≤0.01) were detectable in both smokers and nonsmokers. In smokers, there were no significant changes in sCD40L and sP-selectin but there was a significant increase in platelet aggregation. In nonsmokers, there was a significant increase in all markers of platelet activation following both cigarette and E-cigarette use. Both traditional and E-cigarettes have short-term effects on platelet activation, although in nonsmokers the use of E-cigarettes had a less important impact. In conclusion, we provide the first comparison data of the acute impact of Tobacco-cigarette and E-cigarette smoking on the platelet function in smokers and nonsmokers.

The identification of the mechanisms predisposing to stroke may improve its preventive and therapeutic strategies in patients with essential hypertension. The role of macroautophagy/autophagy in the development of hypertension-related stroke needs to be clarified. We hypothesized that a defective autophagy may favor hypertension-related spontaneous stroke by promoting mitochondrial dysfunction. We studied autophagy in the stroke-prone spontaneously hypertensive (SHRSP) rat, which represents a clinically relevant model of stroke associated with high blood pressure. We assessed autophagy, mitophagy and NAD+:NADH levels in brains of SHRSP and stroke-resistant SHR fed with high salt diet. Vascular smooth muscle cells silenced for the mitochondrial complex I subunit Ndufc2 gene (NADH:ubiquinone oxidoreductase subunit C2) and cerebral endothelial cells isolated from SHRSP were also used to assess autophagy/mitophagy and mitochondrial function in response to high salt levels. We found a reduction of autophagy in brains of high salt-fed SHRSP. Autophagy impairment was associated with NDUFC2 downregulation, mitochondrial dysfunction and NAD+ depletion. Restoration of NAD+ levels by nicotinamide administration reactivated autophagy and reduced stroke development in SHRSP. A selective reactivation of autophagy/mitophagy by Tat-Beclin 1 also reduced stroke occurrence, restored autophagy/mitophagy and improved mitochondrial function. Endothelial progenitor cells (EPCs) from subjects homozygous for the thymine allele variant at NDUFC2/rs11237379, which is associated with NDUFC2 deficiency and increased stroke risk, displayed an impairment of autophagy and increased senescence in response to high salt levels. EPC senescence was rescued by Tat-Beclin 1. Pharmacological activation of autophagy may represent a novel therapeutic strategy to reduce stroke occurrence in hypertension.Abbreviations 10 VSMCs: aortic vascular smooth muscle cells; COX4I1/COX IV: cytochrome c oxidase subunit 4I1; ECs: endothelial cells; EPCs: endothelial progenitor cells; JD: Japanese-style diet; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; NAD: nicotinamide adenine dinucleotide; NDUFC2: NADH:ubiquinone oxidoreductase subunit C2; NMN: nicotinamide mononucleotide; RD: regular diet; SHRSP: stroke-prone spontaneously hypertensive rat; SHRSR: stroke-resistant spontaneously hypertensive rat.

Cardiovascular disease is the leading cause of death in western countries. Among cardiovascular diseases, myocardial infarction represents a life-threatening condition predisposing to the development of heart failure. In recent decades, much effort has been invested in studying the molecular mechanisms underlying the development and progression of ischemia/reperfusion (I/R) injury and post-ischemic cardiac remodeling. These mechanisms include metabolic alterations, ROS overproduction, inflammation, autophagy deregulation and mitochondrial dysfunction. This review article discusses the most recent evidence regarding the molecular basis of myocardial ischemic injury and the new potential therapeutic interventions for boosting cardioprotection and attenuating cardiac remodeling.

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.

SARS-CoV-2 is associated with an increased risk of venous and arterial thrombosis, but the underlying mechanism is still unclear.We performed a cross-sectional analysis of platelet function in 25 SARS-CoV-2 and 10 healthy subjects by measuring Nox2 (NADPH oxidase 2)-derived oxidative stress and thromboxane B2, and investigated if administration of monoclonal antibodies against the S protein (Spike protein) of SARS-CoV-2 affects platelet activation. Furthermore, we investigated in vitro if the S protein of SARS-CoV-2 or plasma from SARS-CoV-2 enhanced platelet activation.Ex vivo studies showed enhanced platelet Nox2-derived oxidative stress and thromboxane B2 biosynthesis and under laminar flow platelet-dependent thrombus growth in SARS-CoV-2 compared with controls; both effects were lowered by Nox2 and TLR4 (Toll-like receptor 4) inhibitors. Two hours after administration of monoclonal antibodies, a significant inhibition of platelet activation was observed in patients with SARS-CoV-2 compared with untreated ones. In vitro study showed that S protein per se did not elicit platelet activation but amplified the platelet response to subthreshold concentrations of agonists and functionally interacted with platelet TLR4. A docking simulation analysis suggested that TLR4 binds to S protein via three receptor-binding domains; furthermore, immunoprecipitation and immunofluorescence showed S protein-TLR4 colocalization in platelets from SARS-CoV-2. Plasma from patients with SARS-CoV-2 enhanced platelet activation and Nox2-related oxidative stress, an effect blunted by TNF (tumor necrosis factor) α inhibitor; this effect was recapitulated by an in vitro study documenting that TNFα alone promoted platelet activation and amplified the platelet response to S protein via p47phox (phagocyte oxidase) upregulation.The study identifies 2 TLR4-dependent and independent pathways promoting platelet-dependent thrombus growth and suggests inhibition of TLR4. or p47phox as a tool to counteract thrombosis in SARS-CoV-2.

Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) is a multisubunit enzyme complex that participates in the generation of superoxide or hydrogen peroxide (H2O2) and plays a key role in several biological functions. Among seven known NOX isoforms, NOX2 was the first identified in phagocytes but is also expressed in several other cell types including endothelial cells, platelets, microglia, neurons, and muscle cells. NOX2 has been assigned multiple roles in regulating many aspects of innate and adaptive immunity, and human and mouse models of NOX2 genetic deletion highlighted this key role. On the other side, NOX2 hyperactivation is involved in the pathogenesis of several diseases with different etiologies but all are characterized by an increase in oxidative stress and inflammatory process. From this point of view, the modulation of NOX2 represents an important therapeutic strategy aimed at reducing the damage associated with its hyperactivation. Although pharmacological strategies to selectively modulate NOX2 are implemented thanks to new biotechnologies, this field of research remains to be explored. Therefore, in this review, we analyzed the role of NOX2 at the crossroads between immunity and pathologies mediated by its hyperactivation. We described (1) the mechanisms of activation and regulation, (2) human, mouse, and cellular models studied to understand the role of NOX2 as an enzyme of innate immunity, (3) some of the pathologies associated with its hyperactivation, and (4) the inhibitory strategies, with reference to the most recent discoveries.

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.

Background Heart failure represents a major cause of disease burden worldwide and is expected to further rise in the coming decades. Hypertension is the clinical condition most frequently associated to heart failure. Objective To systematically review the incidence of heart failure compared to coronary heart diseases and stroke in recent hypertension trials. Methods We identified 23 trials concluded within the last decade including 193 424 patients with hypertension or at ‘high’ cardiovascular risk with a predominant presence of hypertensive patients, and reported incidence of major cardiovascular events, including heart failure, coronary heart disease and stroke. Results A total of 24 837 major cardiovascular events were recorded in trials performed between 1997 and 2007, of which 7171 (28.9%) were cases of heart failure, 10 223 (41.1%) of coronary heart disease and 7443 (30.0%) of stroke. The rate of heart failure was comparable with that of stroke, accounting for 8.5 and 9.1 events per 1000 patients (P = NS), respectively. Heart failure development was more prevalent in older subjects (>65 years) [odds ratio: 3.08, confidence interval 95% (2.88–3.31); P < 0.0001], in black versus nonblack individuals [odds ratio 1.90, (1.76–2.06); P < 0.0001], in diabetic versus nondiabetic patients [odds ratio 4.91, 95% confidence interval (4.40–5.43); P < 0.0001] and in patients with ‘very high’ risk versus those with a ‘high’ risk profile [odds ratio 1.29, 95% confidence interval (1.23–1.36); P < 0.0001]. Conclusion Our analysis shows that heart failure development remains a major problem in hypertension. In recent trials on hypertension, the development of heart failure was found comparable with that of stroke: it is more prevalent in older, black, diabetic and ‘very high’ risk individuals. These findings highlight the relevance of heart failure development in hypertension and support the need for optimizing antihypertensive strategies aimed at preventing the progression to overt heart failure, thus reducing the growing burden of disease associated with hypertension.

BackgroundAngiotensin-converting enzyme (ACE) inhibitors confer renal protection in different clinical settings. No final conclusions are available on the renal benefits of ACE inhibitors after coronary artery bypass grafting (CABG). Because ACE inhibitors decrease glomerular perfusion pressure, they may exacerbate kidney injury during cardiopulmonary bypass (CPB)-related hypoperfusion. We evaluated the effect of preoperative ACE inhibitors on acute kidney injury (AKI) after CABG.MethodsA propensity score–based analysis of 536 patients undergoing CABG on CPB was performed, among which 281 received ACE inhibitors preoperatively. Patients with preoperative end-stage renal failure requiring dialysis were excluded. Postoperative AKI was defined as 50% or more decrease in the glomerular filtration rate from preoperative or postoperative mechanical renal support.ResultsAfter CABG, AKI developed in 49 patients (9.1%), and 23 (4.2%) required dialysis. The incidence of AKI was 6.4% in patients who received preoperative ACE inhibitors and 12.2% in patients who did not (p = 0.02). The incidence of AKI requiring dialysis was 2.4% in the treatment group and 6.3% in controls (p = 0.03). After adjusting for propensity score and covariates, preoperative ACE inhibitors were found to reduce the incidence of postoperative AKI (odds ratio, 0.48; 95% confidence interval, 0.23 to 0.77; p = 0.04). Other independent predictors were age, preoperative glomerular filtration rate, left ventricular ejection fraction of less than 0.35, preoperative use of intraaortic balloon pump, emergency operation, and CPB time.ConclusionsPreoperative ACE inhibitors are associated with a reduced rate of AKI after on-pump CABG surgery. Angiotensin-converting enzyme (ACE) inhibitors confer renal protection in different clinical settings. No final conclusions are available on the renal benefits of ACE inhibitors after coronary artery bypass grafting (CABG). Because ACE inhibitors decrease glomerular perfusion pressure, they may exacerbate kidney injury during cardiopulmonary bypass (CPB)-related hypoperfusion. We evaluated the effect of preoperative ACE inhibitors on acute kidney injury (AKI) after CABG. A propensity score–based analysis of 536 patients undergoing CABG on CPB was performed, among which 281 received ACE inhibitors preoperatively. Patients with preoperative end-stage renal failure requiring dialysis were excluded. Postoperative AKI was defined as 50% or more decrease in the glomerular filtration rate from preoperative or postoperative mechanical renal support. After CABG, AKI developed in 49 patients (9.1%), and 23 (4.2%) required dialysis. The incidence of AKI was 6.4% in patients who received preoperative ACE inhibitors and 12.2% in patients who did not (p = 0.02). The incidence of AKI requiring dialysis was 2.4% in the treatment group and 6.3% in controls (p = 0.03). After adjusting for propensity score and covariates, preoperative ACE inhibitors were found to reduce the incidence of postoperative AKI (odds ratio, 0.48; 95% confidence interval, 0.23 to 0.77; p = 0.04). Other independent predictors were age, preoperative glomerular filtration rate, left ventricular ejection fraction of less than 0.35, preoperative use of intraaortic balloon pump, emergency operation, and CPB time. Preoperative ACE inhibitors are associated with a reduced rate of AKI after on-pump CABG surgery.

&lt;i&gt;Background:&lt;/i&gt; While chronic dialysis treatment has been suggested to increase pulmonary pressure values, right ventricular dysfunction (RVD) is a major cause of death in patients with end-stage renal disease. We investigated the impact of different dialysis treatments on right ventricular function. &lt;i&gt;Methods:&lt;/i&gt; We examined 220 subjects grouped as follows: healthy controls (n = 100), peritoneal dialysis (PD; n = 26), hemodialysis (HD) with radial arteriovenous fistula (AVF; n = 62), and HD with brachial AVF (n = 32). Echocardiography including tissue Doppler imaging (TDI) of the right ventricle was performed in all patients. &lt;i&gt;Results:&lt;/i&gt; Pulmonary pressure values progressively rose from controls across the 3 dialysis groups (21.7 ± 6.8, 29.7 ± 6.7, 37.9 ± 6.7 and 40.8 ± 6.6 mm Hg, respectively; p &lt; 0.001). TDI indices of right ventricular function were more impaired in HD patients, particularly in those with brachial AVF. RVD, assessed by TDI myocardial performance index, was higher in HD patients compared with PD patients (71.3 vs. 34.6%, p &lt; 0.001). Moreover, the prevalence of RVD further increased in patients with brachial AVF compared with the radial access (90.6 vs. 61.3%, p &lt; 0.001). &lt;i&gt;Conclusions:&lt;/i&gt; Compared to DP, HD increases the risk of RVD, particularly in the presence of brachial AVF. TDI may detect early functional failure of the right ventricle in HD patients.

To determine whether the administration of angiotensin-converting enzyme (ACE) inhibitors or angiotensin II receptor blockers (ARBs) on top of standard cardiovascular (CV) therapies may reduce the incidence of new onset diabetes (NOD) in placebo-controlled clinical trials. The effects of these drugs on CV and non-CV mortality were also tested.We performed a meta-analysis of all randomized clinical trials (11 trials, n = 84,363 patients, aged 64.2 ± 5.86 years), published until 14 March 2010, in which ACE inhibitors or ARBs were compared with placebo and NOD incidence, CV, and non-CV mortality were reported.Over an average follow-up of 4.0 ± 1.0 years, there were 1,284/15,142 (8.5%) cases of NOD in active-treated and 1,411/15,130 (9.3%) cases in placebo-treated patients in the ACE inhibitor trials, and 2,330/18,756 (12.4%) cases in active-treated and 2,669/18,800 (14.2%) cases in placebo-treated patients in the ARB trials. Overall, active therapy reduced NOD compared to placebo (odds ratio (OR) 95%, confidence interval (CI): 0.8 (0.8-0.9); P < 0.01). Both ACE inhibitors (OR 95%, CI: 0.8 (0.7-1.0); P = 0.07) and ARBs (OR 95%, CI: 0.8 (0.8-0.9); P < 0.01) reduced NOD as compared to placebo. Active treatment reduced CV mortality (OR 95%, CI: 0.9 (0.8-1.0); P < 0.01) and had a favorable impact on non-CV mortality (OR 95%, CI: 0.7 (0.9-1.0); P = 0.2) as compared to placebo.Our findings demonstrated that ACE inhibitors or ARBs should be preferred in patients with clinical conditions that may increase risk of NOD, since these drugs reduced NOD incidence. In addition, these drugs have favorable effects on CV and non-CV mortality in high CV risk patients.

The Rag family proteins are Ras-like small GTPases that have a critical role in amino-acid-stimulated mTORC1 activation by recruiting mTORC1 to lysosome. Despite progress in the mechanistic understanding of Rag GTPases in mTORC1 activation, little is known about the physiological function of Rag GTPases in vivo. Here we show that loss of RagA and RagB (RagA/B) in cardiomyocytes results in hypertrophic cardiomyopathy and phenocopies lysosomal storage diseases, although mTORC1 activity is not substantially impaired in vivo. We demonstrate that despite upregulation of lysosomal protein expression by constitutive activation of the transcription factor EB (TFEB) in RagA/B knockout mouse embryonic fibroblasts, lysosomal acidification is compromised owing to decreased v-ATPase level in the lysosome fraction. Our study uncovers RagA/B GTPases as key regulators of lysosomal function and cardiac protection.

Data regarding coronary artery calcification (CAC) prognosis in diabetic individuals are limited to 5-years follow-up. We investigated the long-term risk stratification of CAC among diabetic compared with nondiabetic individuals.Nine thousand seven hundred and fifteen asymptomatic individuals undergoing CAC scoring were followed for a median (interquartile range) of 14.7 (13.9-15.6) years. The incidence density rate and hazard ratios with 95% confidence intervals were used to calculate all-cause mortality. Incremental prognostic utility of CAC was evaluated using the area under the receiver operator characteristic curve and net reclassification improvement. Diabetics (54.7±10.8 years; 59.4% male) comprised 8.3% of the cohort (n=810), of which 188 (23.2%) died. For CAC=0, the rate of mortality was similar between diabetic and nondiabetic individuals for the first 5 years (P>0.05), with a nonlinear increased risk of mortality for diabetics after 5 years (P<0.05). The adjusted risk of death for those in the highest (CAC>400) versus the lowest (CAC=0) category of CAC increased by a hazards of 4.64 (95% confidence interval =3.74-5.76) and 3.41 (95% confidence interval =2.22-5.22) for nondiabetic and diabetic individuals, respectively. The presence of CAC improved discrimination (area under the receiver operator characteristic curve range: 0.73-0.74; P<0.01) and reclassification (category-free net reclassification improvement range: 0.53-0.50; P<0.001) beyond conventional risk factors in nondiabetic and diabetic individuals, respectively.CAC=0 is associated with a favorable 5-year prognosis for asymptomatic diabetic and nondiabetic individuals. After 5 years, the risk of mortality increases significantly for diabetic individuals even in the presence of a baseline CAC=0.

Reactive oxygen species (ROS) may cause cellular damage and oxidative stress-induced cell death. Autophagy, an evolutionarily conserved intracellular catabolic process, is executed by autophagy (ATG) proteins, including the autophagy initiation kinase Unc-51-like kinase (ULK1)/ATG1. Although autophagy has been implicated to have both cytoprotective and cytotoxic roles in the response to ROS, the role of individual ATG proteins, including ULK1, remains poorly characterized. In this study, we demonstrate that ULK1 sensitizes cells to necrotic cell death induced by hydrogen peroxide (H2O2). Moreover, we demonstrate that ULK1 localizes to the nucleus and regulates the activity of the DNA damage repair protein poly (ADP-ribose) polymerase 1 (PARP1) in a kinase-dependent manner. By enhancing PARP1 activity, ULK1 contributes to ATP depletion and death of H2O2-treated cells. Our study provides the first evidence of an autophagy-independent prodeath role for nuclear ULK1 in response to ROS-induced damage. On the basis of our data, we propose that the subcellular distribution of ULK1 has an important role in deciding whether a cell lives or dies on exposure to adverse environmental or intracellular conditions.

Diabetes mellitus is a common disease that affects 3⁻5% of the general population in Italy. In some countries of northern Europe or in North America, it can even affect 6⁻8% of the population. Of great concern is that the number of cases of diabetes is constantly increasing, probably due to the increase in obesity and the sedentary nature of the population. According to the World Health Organization, in the year 2030 there will be 360 million people with diabetes, compared to 170 million in 2000. This has important repercussions on the lives of patients and their families, and on health systems that offer assistance to patients. In this review, we try to describe in an organized way the pathophysiological continuity between diabetes mellitus, endothelial dysfunction, and platelet hyperaggregation, highlighting the main molecular mechanisms involved and the interconnections.

Atrial natriuretic peptide (ANP) is a cardiac hormone belonging to the family of natriuretic peptides (NPs). ANP exerts diuretic, natriuretic, and vasodilatory effects that contribute to maintain water-salt balance and regulate blood pressure. Besides these systemic properties, ANP displays important pleiotropic effects in the heart and in the vascular system that are independent of blood pressure regulation. These functions occur through autocrine and paracrine mechanisms. Previous works examining the cardiac phenotype of loss-of-function mouse models of ANP signaling showed that both mice with gene deletion of ANP or its receptor natriuretic peptide receptor A (NPR-A) developed cardiac hypertrophy and dysfunction in response to pressure overload and chronic ischemic remodeling. Conversely, ANP administration has been shown to improve cardiac function in response to remodeling and reduces ischemia-reperfusion (I/R) injury. ANP also acts as a pro-angiogenetic, anti-inflammatory, and anti-atherosclerotic factor in the vascular system. Pleiotropic effects regarding brain natriuretic peptide (BNP) and C-type natriuretic peptide (CNP) were also reported. In this review, we discuss the current evidence underlying the pleiotropic effects of NPs, underlying their importance in cardiovascular homeostasis.

Considered as a superfood of the future, Spirulina platensis matrix has been extensively used because of its beneficial effect on the management of cardiovascular diseases. However, its nutraceutical properties, bioactive compounds, and molecular mechanisms are unknown. Here, we demonstrate that S platensis matrix processed in vitro by simulated gastrointestinal digestion induces direct endothelial nitric oxide (NO)-mediated vasorelaxation of resistance vessels in mice. To gain insight into the bioactive compounds responsible for this effect, we used a complex multistep peptidomic approach to fractionate the crude digest: of the 5 peptide fractions identified (A-E), only fraction E evoked vasorelaxation. High-resolution mass spectrometry-based screening revealed in E the presence of 4 main peptides (SP3-SP6 [spirulina peptides]), of which only SP6 (GIVAGDVTPI) exerted direct endothelium-dependent vasodilation of ex vivo vessels, an effect occurring via a PI3K (phosphoinositide-3-kinase)/AKT (serine/threonine kinase Akt) pathway converging on NO release. In vivo, administration of SP6 evoked a significant hemodynamic effect, reducing blood pressure, an action absent in eNOS (endothelial NO synthase)-deficient mice. Of note, although lower doses of SP6 had no hemodynamic effects, it still enhanced endothelial NO vasorelaxation. Finally, in an experimental model of arterial hypertension, SP6 exerted an antihypertensive effect, improving endothelial vasorelaxation associated with enhanced serum nitrite levels. Based on our results, this novel decameric peptide may extend the possible fields of application for spirulina-derived peptides and could be developed into a promising nonpharmacological approach for the containment of pathologies associated with vascular NO misregulation.

Here, we aimed to determine the therapeutic effect of longevity-associated variant (LAV)-BPIFB4 gene therapy on atherosclerosis.ApoE knockout mice (ApoE-/-) fed a high-fat diet were randomly allocated to receive LAV-BPIFB4, wild-type (WT)-BPIFB4, or empty vector via adeno-associated viral vector injection. The primary endpoints of the study were to assess (i) vascular reactivity and (ii) atherosclerotic disease severity, by Echo-Doppler imaging, histology and ultrastructural analysis. Moreover, we assessed the capacity of the LAV-BPIFB4 protein to shift monocyte-derived macrophages of atherosclerotic mice and patients towards an anti-inflammatory phenotype. LAV-BPIFB4 gene therapy rescued endothelial function of mesenteric and femoral arteries from ApoE-/- mice; this effect was blunted by AMD3100, a CXC chemokine receptor type 4 (CXCR4) inhibitor. LAV-BPIFB4-treated mice showed a CXCR4-mediated shift in the balance between Ly6Chigh/Ly6Clow monocytes and M2/M1 macrophages, along with decreased T cell proliferation and elevated circulating levels of interleukins IL-23 and IL-27. In vitro conditioning with LAV-BPIFB4 protein of macrophages from atherosclerotic patients resulted in a CXCR4-dependent M2 polarization phenotype. Furthermore, LAV-BPIFB4 treatment of arteries explanted from atherosclerotic patients increased the release of atheroprotective IL-33, while inhibiting the release of pro-inflammatory IL-1β, inducing endothelial nitric oxide synthase phosphorylation and restoring endothelial function. Finally, significantly lower plasma BPIFB4 was detected in patients with pathological carotid stenosis (>25%) and intima media thickness >2 mm.Transfer of the LAV of BPIFB4 reduces the atherogenic process and skews macrophages towards an M2-resolving phenotype through modulation of CXCR4, thus opening up novel therapeutic possibilities in cardiovascular disease.

Abstract Bicuspid aortic valve (BAV) is frequently associated with the development of ascending aortic aneurysm, even if the underlying mechanisms remain to be clarified. Here, we investigated if a deregulation of Notch1 signaling pathway and endothelial progenitor cells (EPCs) number is associated with BAV disease and an early ascending aortic aneurysm (AAA) onset. For this purpose, 70 subjects with BAV (M/F 50/20; mean age: 58.8 ± 14.8 years) and 70 subjects with tricuspid aortic valve (TAV) (M/F 35/35; mean age: 69.1 ± 12.8 years) and AAA complicated or not, were included. Interestingly, patients with AAA showed a significant increase in circulating Notch1 levels and EPC number than subjects without AAA. However, circulating Notch1 levels and EPC number were significantly lower in BAV subjects than TAV patients either in the presence or absence of AAA. Finally, Notch pathway was activated to a greater extent in aortic aneurysmatic portions with respect to healthy aortic fragments in both BAV and TAV patients. However, the expression of genes encoding components and ligands of Notch pathway in aortic tissues was significantly lower in BAV than TAV subjects. Our study demonstrates that BAV subjects are characterized by a significant decrease in both tissue and circulating levels of Notch pathway, and in blood EPC number than TAV patients, either in presence or absence of AAA disease.

Oxidative stress may be defined as an imbalance between reactive oxygen species (ROS) and the antioxidant system to counteract or detoxify these potentially damaging molecules. This phenomenon is a common feature of many human disorders, such as cardiovascular disease. Many of the risk factors, including smoking, hypertension, hypercholesterolemia, diabetes, and obesity, are associated with an increased risk of developing cardiovascular disease, involving an elevated oxidative stress burden (either due to enhanced ROS production or decreased antioxidant protection). There are many therapeutic options to treat oxidative stress-associated cardiovascular diseases. Numerous studies have focused on the utility of antioxidant supplementation. However, whether antioxidant supplementation has any preventive and/or therapeutic value in cardiovascular pathology is still a matter of debate. In this review, we provide a detailed description of oxidative stress biomarkers in several cardiovascular risk factors. We also discuss the clinical implications of the supplementation with several classes of antioxidants, and their potential role for protecting against cardiovascular risk factors.

Lysosomal dysfunction caused by mutations in lysosomal genes results in lysosomal storage disorder (LSD), characterized by accumulation of damaged proteins and organelles in cells and functional abnormalities in major organs, including the heart, skeletal muscle, and liver. In LSD, autophagy is inhibited at the lysosomal degradation step and accumulation of autophagosomes is observed. Enlargement of the left ventricle (LV) and contractile dysfunction were observed in RagA/B cardiac-specific KO (cKO) mice, a mouse model of LSD in which lysosomal acidification is impaired irreversibly. YAP, a downstream effector of the Hippo pathway, was accumulated in RagA/B cKO mouse hearts. Inhibition of YAP ameliorated cardiac hypertrophy and contractile dysfunction and attenuated accumulation of autophagosomes without affecting lysosomal function, suggesting that YAP plays an important role in mediating cardiomyopathy in RagA/B cKO mice. Cardiomyopathy was also alleviated by downregulation of Atg7, an intervention to inhibit autophagy, whereas it was exacerbated by stimulation of autophagy. YAP physically interacted with transcription factor EB (TFEB), a master transcription factor that controls autophagic and lysosomal gene expression, thereby facilitating accumulation of autophagosomes without degradation. These results indicate that accumulation of YAP in the presence of LSD promotes cardiomyopathy by stimulating accumulation of autophagosomes through activation of TFEB.

Aims: Therapeutic modulation of blood vessel growth holds promise for the prevention of limb ischemia in diabetic (DM) patients with peripheral artery disease (PAD). Epigenetic changes, namely, posttranslational histone modifications, participate in angiogenic response suggesting that chromatin-modifying drugs could be beneficial in this setting. Apabetalone (APA), a selective inhibitor of bromodomain (BRD) and bromodomain and extraterminal containing protein family (BET) proteins, prevents bromodomain-containing protein 4 (BRD4) interactions with chromatin thus modulating transcriptional programs in different organs. We sought to investigate whether APA affects angiogenic response in diabetes. Results: Compared with vehicle, APA restored tube formation and migration in human aortic endothelial cells (HAECs) exposed to high-glucose (HG) levels. Expression profiling of angiogenesis genes showed that APA prevents HG-induced upregulation of the antiangiogenic molecule thrombospondin-1 (THBS1). ChIP-seq and chromatin immunoprecipitation (ChIP) assays in HG-treated HAECs showed the enrichment of both BRD4 and active marks (H3K27ac) on THBS1 promoter, whereas BRD4 inhibition by APA prevented chromatin accessibility and THBS1 transcription. Mechanistically, we show that THBS1 inhibits angiogenesis by suppressing vascular endothelial growth factor A (VEGFA) signaling, while APA prevents these detrimental changes. In diabetic mice with hind limb ischemia, epigenetic editing by APA restored the THBS1/VEGFA axis, thus improving limb vascularization and perfusion, compared with vehicle-treated animals. Finally, epigenetic regulation of THBS1 by BRD4/H3K27ac was also reported in DM patients with PAD compared with nondiabetic controls. Innovation: This is the first study showing that BET protein inhibition by APA restores angiogenic response in experimental diabetes. Conclusions: Our findings set the stage for preclinical studies and exploratory clinical trials testing APA in diabetic PAD. Antioxid. Redox Signal. 36, 667-684.

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.

Autophagy is an evolutionarily conserved intracellular mechanism for degradation of long-lived proteins and organelles. Accumulating lines of evidence indicate that autophagy is deeply involved in the development of cardiac disease. Autophagy is upregulated in almost all cardiac pathological states, exerting both protective and detrimental functions. Whether autophagy activation is an adaptive or maladaptive mechanism during cardiac stress seems to depend upon the pathological context in which it is upregulated, the extent of its activation, and the signaling mechanisms promoting its enhancement. Pharmacological modulation of autophagy may therefore represent a potential therapeutic strategy to limit myocardial damage during cardiac stress. Several pharmacological agents that are able to modulate autophagy have been identified, such as mammalian target of rapamycin inhibitors, adenosine monophosphate–dependent kinase modulators, sirtuin activators, myo-inositol-1,4,5-triphosphate and calcium-lowering agents, and lysosome inhibitors. Although few of these modulators of autophagy have been directly tested during cardiac stress, many of them seem to have high potential to be efficient in the treatment of cardiac disease. We will discuss the potential usefulness of different pharmacological activators and inhibitors of autophagy in the treatment of cardiac diseases.

Cardiac hypertrophy, an adaptive process that responds to increased wall stress, is characterized by the enlargement of cardiomyocytes and structural remodeling. It is stimulated by various growth signals, of which the mTORC1 pathway is a well-recognized source. Here, we show that loss of Flcn, a novel AMPK-mTOR interacting molecule, causes severe cardiac hypertrophy with deregulated energy homeostasis leading to dilated cardiomyopathy in mice. We found that mTORC1 activity was upregulated in Flcn-deficient hearts, and that rapamycin treatment significantly reduced heart mass and ameliorated cardiac dysfunction. Phospho-AMP-activated protein kinase (AMPK)-alpha (T172) was reduced in Flcn-deficient hearts and nonresponsive to various stimulations including metformin and AICAR (5-amino-1-β-D-ribofuranosyl-imidazole-4-carboxamide). ATP levels were elevated and mitochondrial function was increased in Flcn-deficient hearts, suggesting that excess energy resulting from up-regulated mitochondrial metabolism under Flcn deficiency might attenuate AMPK activation. Expression of Ppargc1a, a central molecule for mitochondrial metabolism, was increased in Flcn-deficient hearts and indeed, inactivation of Ppargc1a in Flcn-deficient hearts significantly reduced heart mass and prolonged survival. Ppargc1a inactivation restored phospho-AMPK-alpha levels and suppressed mTORC1 activity in Flcn-deficient hearts, suggesting that up-regulated Ppargc1a confers increased mitochondrial metabolism and excess energy, leading to inactivation of AMPK and activation of mTORC1. Rapamycin treatment did not affect the heart size of Flcn/Ppargc1a doubly inactivated hearts, further supporting the idea that Ppargc1a is the critical element leading to deregulation of the AMPK-mTOR-axis and resulting in cardiac hypertrophy under Flcn deficiency. These data support an important role for Flcn in cardiac homeostasis in the murine model.

NADPH oxidase is a cellular enzyme devoted to the production of reactive oxygen species (ROS). NOX4 and NOX2 are the main isoforms of NADPH oxidase in the cardiovascular system. In our recent study, we demonstrated that NOX4, but not NOX2, is a critical mediator of the cardiomyocyte adaptive response to energy stress. NOX4 activity and protein levels are increased in the endoplasmic reticulum (ER) but not in mitochondria of cardiomyocytes during the early phase of energy deprivation. NOX4-derived production of ROS in the ER is a critical event that activates autophagy through stimulation of the EIF2AK3/PERK-EIF2S1/eIF-2α-ATF4 pathway. NOX4-dependent autophagy is an important mechanism to preserve cellular energy and limit cell death in energy-deprived cardiomyocytes. Aside from elucidating a crucial physiological function of NOX4 during cellular energy stress, our study dissects a novel signaling mechanism that regulates autophagy under this condition.

The genetic basis of stroke susceptibility remains to be elucidated. STR1 quantitative trait locus (STR1/QTL) was identified on rat chromosome 1 of stroke-prone spontaneously hypertensive rat (SHRSP) upon Japanese-style stroke-permissive diet (JD), and it contributes to 20% of the stroke phenotype variance.Nine hundred eighty-six probe sets mapping on STR1 were selected from the Rat RAE230A array and screened through a microarray differential expression analysis in brains of SHRSP and stroke-resistant SHR (SHRSR) fed with either regular diet or JD. The gene encoding Ndufc2 (NADH dehydrogenase [ubiquinone] 1 subunit), mapping 8 Mb apart from STR1/QTL Lod score peak, was found significantly down-regulated under JD in SHRSP compared to SHRSR. Ndufc2 disruption altered complex I assembly and activity, reduced mitochondrial membrane potential and ATP levels, and increased reactive oxygen species production and inflammation both in vitro and in vivo. SHRSR carrying heterozygous Ndufc2 deletion showed renal abnormalities and stroke occurrence under JD, similarly to SHRSP. In humans, T allele variant at NDUFC2/rs11237379 was associated with significant reduction in gene expression and with increased occurrence of early-onset ischemic stroke by recessive mode of transmission (odds ratio [OR], 1.39; CI, 1.07-1.80; P=0.012). Subjects carrying TT/rs11237379 and A allele variant at NDUFC2/rs641836 had further increased risk of stroke (OR=1.56; CI, 1.14-2.13; P=0.006).A significant reduction of Ndufc2 expression causes complex I dysfunction and contributes to stroke susceptibility in SHRSP. Moreover, our current evidence may suggest that Ndufc2 can contribute to an increased occurrence of early-onset ischemic stroke in humans.

Autophagy is a highly conserved recycling mechanism essential for maintaining cellular homeostasis. The pathophysiological role of autophagy has been explored since its discovery 50 years ago, but interest in autophagy has grown exponentially over the last years. Many researchers around the globe have found that autophagy is a critical pathway involved in the pathogenesis of cardiac diseases. Several groups have created novel and powerful tools for gaining deeper insights into the role of autophagy in the aetiology and development of pathologies affecting the heart. Here, we discuss how established and emerging methods to study autophagy can be used to unravel the precise function of this central recycling mechanism in the cardiac system.

Cardiovascular diseases (CVDs) such as hypertension, atherosclerosis, myocardial infarction, and diabetes are a significant public health problem worldwide. Although several novel pharmacological treatments to reduce the progression of CVDs have been discovered during the last 20 years, the better way to contain the onset of CVDs remains prevention. In this regard, nutraceuticals seem to own a great potential in maintaining human health, exerting important protective cardiovascular effects. In the last years, there has been increased focus on identifying natural compounds with cardiovascular health-promoting effects and also to characterize the molecular mechanisms involved. Although many review articles have focused on the individual natural compound impact on cardiovascular diseases, the aim of this manuscript was to examine the role of the most studied nutraceuticals, such as resveratrol, cocoa, quercetin, curcumin, brassica, berberine and Spirulina platensis, on different CVDs.

The novel coronavirus named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has generated the ongoing coronavirus disease-2019 (COVID-19) pandemic, still with an uncertain outcome. Besides pneumonia and acute lung injury (ALI) or acute respiratory distress syndrome (ARDS), other features became evident in the context of COVID-19. These includes endothelial and coagulation dysfunction with disseminated intravascular coagulation (DIC), and multiple organ dysfunction syndrome (MODS), along with the occurrence of neurological alterations. The multi-system nature of such viral infection is a witness to the exploitation and impairment of ubiquitous subcellular and metabolic pathways for the sake of its life-cycle, ranging from host cell invasion, replication, transmission, up to a cytopathic effect and overt systemic inflammation. In this frame, alterations in cell-clearing systems of the host are emerging as a hallmark in the pathogenesis of various respiratory viruses, including SARS-CoV-2. Indeed, exploitation of the autophagy and proteasome pathways might contribute not only to the replication of the virus at the site of infection but also to the spreading of either mature virions or inflammatory mediators at both cellular and multisystem levels. In this frame, besides a pharmacological therapy, many researchers are wondering if some non-pharmacological substances might counteract or positively modulate the course of the infection. The pharmacological properties of natural compounds have gained increasing attention in the field of alternative and adjunct therapeutic approaches to several diseases. In particular, several naturally-occurring herbal compounds (mostly polyphenols) are reported to produce widespread antiviral, anti-inflammatory, and anti-oxidant effects while acting as autophagy and (immuno)-proteasome modulators. This article attempts to bridge the perturbation of autophagy and proteasome pathways with the potentially beneficial effects of specific phytochemicals and flavonoids in viral infections, with a focus on the multisystem SARS-CoV-2 infection.

The mechanistic target of rapamycin (mTOR) integrates several intracellular and extracellular signals involved in the regulation of anabolic and catabolic processes. mTOR assembles into two macromolecular complexes, named mTORC1 and mTORC2, which have different regulators, substrates and functions. Studies of gain- and loss-of-function animal models of mTOR signalling revealed that mTORC1/2 elicits both adaptive and maladaptive functions in the cardiovascular system. Both mTORC1 and mTORC2 are indispensable for driving cardiac development and cardiac adaption to stress, such as pressure overload. However, persistent and deregulated mTORC1 activation in the heart is detrimental during stress and contributes to the development and progression of cardiac remodelling and genetic and metabolic cardiomyopathies. In this review, we discuss the latest findings regarding the role of mTOR in the cardiovascular system, both under basal conditions and during stress, such as pressure overload, ischemia, and metabolic stress. Current data suggest that mTOR modulation may represent a potential therapeutic strategy for the treatment of cardiac diseases.

Tobacco habit still represents the leading preventable cause of morbidity and mortality worldwide. Heat-not-burn cigarettes (HNBCs) are considered as an alternative to traditional combustion cigarettes (TCCs) due to the lack of combustion and the absence of combustion-related specific toxicants. The aim of this observational study was to assess the effect of HNBC on endothelial function, oxidative stress and platelet activation in chronic adult TCC smokers and HNBC users. The results showed that both HNBC and TCC display an adverse phenotype in terms of endothelial function, oxidative stress and platelet activation. Future randomised studies are strongly warranted to confirm these data.

Beyond well-assessed risk factors, cardiovascular events could be also associated with the presence of epigenetic and genetic alterations, such as the methylenetetrahydrofolate-reductase (MTHFR) C677T polymorphism. This gene variant is related to increased circulating levels of homocysteine (Hcy) and cardiovascular risk. However, heterozygous carriers have an augmented risk of cardiovascular accidents independently from normal Hcy levels, suggesting the presence of additional deregulated processes in MTHFR C677T carriers. Here, we hypothesize that targeting Sirtuin 1 (SIRT1) could be an alternative mechanism to control the cardiovascular risk associated to MTHFR deficiency condition. Flow Mediated Dilatation (FMD) and light transmission aggregometry assay were performed in subjects carrying MTHFR C677T allele after administration of resveratrol, the most powerful natural clinical usable compound that owns SIRT1 activating properties. MTHFR C677T carriers with normal Hcy levels revealed endothelial dysfunction and enhanced platelet aggregation associated with SIRT1 downregulation. SIRT1 activity stimulation by resveratrol intake was able to override these abnormalities without affecting Hcy levels. Impaired endothelial function, bleeding time, and wire-induced thrombus formation were rescued in a heterozygous Mthfr-deficient (Mthfr+/-) mouse model after resveratrol treatment. Using a cell-based high-throughput multiplexed screening (HTS) assay, a novel selective synthetic SIRT1 activator, namely ISIDE11, was identified. Ex vivo and in vivo treatment of Mthfr+/- mice with ISIDE11 rescues endothelial vasorelaxation and reduces wire-induced thrombus formation, effects that were abolished by SIRT1 inhibitor. Moreover, platelets from MTHFR C677T allele carriers treated with ISIDE11 showed normalization of their typical hyper-reactivity. These results candidate SIRT1 activation as a new therapeutic strategy to contain cardio and cerebrovascular events in MTHFR carriers.

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.

The aim of this study was to investigate the impact of rs5065 atrial natriuretic peptide (ANP) gene variant on coronary artery disease (CAD) and its outcomes and to gain potential mechanistic insights on the association with CAD. Either modified ANP plasma levels or peptide structural alterations have been involved in development of cardiovascular events. Three hundred ninety-three control subjects and 1,004 patients undergoing coronary angiography for suspected CAD (432 stable angina [SA], 572 acute coronary syndrome [ACS]) were genotyped for rs5065 ANP gene variant. Data in SA and ACS groups were replicated in an independent population of 482 stable angina patients (rSA) and of 675 ACS patients, respectively. Clinical follow-up was available for both SA and rSA patients. Plasma N-terminal-proANP, myeloperoxidase, lipoprotein-associated phospholipase A2, and oxidized low-density lipoprotein were assessed in a subgroup of rSA patients. rs5065 minor allele (MA) was an independent predictor of ACS (odds ratio: 1.90; 95% confidence interval: 1.40 to 2.58, p < 0.001). At follow-up, rs5065 MA was independently associated with a significantly higher rate of major adverse cardiovascular events in the SA group, p < 0.001. Data were replicated in the rSA group at follow-up (p = 0.008). Cox proportional hazard analysis tested by 4 models confirmed higher major adverse cardiovascular events risk in rs5065 MA carriers in both SA and rSA cohorts. Significantly higher myeloperoxidase levels were detected in rs5065 MA carriers (n = 597 [345 to 832 μg/l] vs. n = 488 [353 to 612 μg/l], p = 0.038). No association of rs5065 was observed with N-terminal-proANP levels. The MA of rs5065 ANP gene variant associates with increased susceptibility to ACS and has unfavorable prognostic value in CAD.

Thioredoxin 1 (Trx1) is a 12-kDa oxidoreductase that catalyzes thiol-disulfide exchange reactions to reduce proteins with disulfide bonds. As such, Trx1 helps protect the heart against stresses, such as ischemia and pressure overload. Mechanistic target of rapamycin (mTOR) is a serine/threonine kinase that regulates cell growth, metabolism, and survival. We have shown previously that mTOR activity is increased in response to myocardial ischemia–reperfusion injury. However, whether Trx1 interacts with mTOR to preserve heart function remains unknown. Using a substrate-trapping mutant of Trx1 (Trx1C35S), we show here that mTOR is a direct interacting partner of Trx1 in the heart. In response to H2O2 treatment in cardiomyocytes, mTOR exhibited a high molecular weight shift in non-reducing SDS-PAGE in a 2-mercaptoethanol-sensitive manner, suggesting that mTOR is oxidized and forms disulfide bonds with itself or other proteins. The mTOR oxidation was accompanied by reduced phosphorylation of endogenous substrates, such as S6 kinase (S6K) and 4E-binding protein 1 (4E-BP1) in cardiomyocytes. Immune complex kinase assays disclosed that H2O2 treatment diminished mTOR kinase activity, indicating that mTOR is inhibited by oxidation. Of note, Trx1 overexpression attenuated both H2O2-mediated mTOR oxidation and inhibition, whereas Trx1 knockdown increased mTOR oxidation and inhibition. Moreover, Trx1 normalized H2O2-induced down-regulation of metabolic genes and stimulation of cell death, and an mTOR inhibitor abolished Trx1-mediated rescue of gene expression. H2O2-induced oxidation and inhibition of mTOR were attenuated when Cys-1483 of mTOR was mutated to phenylalanine. These results suggest that Trx1 protects cardiomyocytes against stress by reducing mTOR at Cys-1483, thereby preserving the activity of mTOR and inhibiting cell death. Thioredoxin 1 (Trx1) is a 12-kDa oxidoreductase that catalyzes thiol-disulfide exchange reactions to reduce proteins with disulfide bonds. As such, Trx1 helps protect the heart against stresses, such as ischemia and pressure overload. Mechanistic target of rapamycin (mTOR) is a serine/threonine kinase that regulates cell growth, metabolism, and survival. We have shown previously that mTOR activity is increased in response to myocardial ischemia–reperfusion injury. However, whether Trx1 interacts with mTOR to preserve heart function remains unknown. Using a substrate-trapping mutant of Trx1 (Trx1C35S), we show here that mTOR is a direct interacting partner of Trx1 in the heart. In response to H2O2 treatment in cardiomyocytes, mTOR exhibited a high molecular weight shift in non-reducing SDS-PAGE in a 2-mercaptoethanol-sensitive manner, suggesting that mTOR is oxidized and forms disulfide bonds with itself or other proteins. The mTOR oxidation was accompanied by reduced phosphorylation of endogenous substrates, such as S6 kinase (S6K) and 4E-binding protein 1 (4E-BP1) in cardiomyocytes. Immune complex kinase assays disclosed that H2O2 treatment diminished mTOR kinase activity, indicating that mTOR is inhibited by oxidation. Of note, Trx1 overexpression attenuated both H2O2-mediated mTOR oxidation and inhibition, whereas Trx1 knockdown increased mTOR oxidation and inhibition. Moreover, Trx1 normalized H2O2-induced down-regulation of metabolic genes and stimulation of cell death, and an mTOR inhibitor abolished Trx1-mediated rescue of gene expression. H2O2-induced oxidation and inhibition of mTOR were attenuated when Cys-1483 of mTOR was mutated to phenylalanine. These results suggest that Trx1 protects cardiomyocytes against stress by reducing mTOR at Cys-1483, thereby preserving the activity of mTOR and inhibiting cell death.

Diabetes, obesity, and dyslipidemia are main risk factors that promote the development of cardiovascular diseases. These metabolic abnormalities are frequently found to be associated together in a highly morbid clinical condition called metabolic syndrome. Metabolic derangements promote endothelial dysfunction, atherosclerotic plaque formation and rupture, cardiac remodeling and dysfunction. This evidence strongly encourages the elucidation of the mechanisms through which obesity, diabetes, and metabolic syndrome induce cellular abnormalities and dysfunction in order to discover new therapeutic targets and strategies for their prevention and treatment. Numerous studies employing both dietary and genetic animal models of obesity and diabetes have demonstrated that autophagy, an intracellular system for protein degradation, is impaired in the heart under these conditions. This suggests that autophagy reactivation may represent a future potential therapeutic intervention to reduce cardiac maladaptive alterations in patients with metabolic derangements. In fact, autophagy is a critical mechanism to preserve cellular homeostasis and survival. In addition, the physiological activation of autophagy protects the heart during stress, such as acute ischemia, starvation, chronic myocardial infarction, pressure overload, and proteotoxic stress. All these aspects will be discussed in our review article together with the potential ways to reactivate autophagy in the context of obesity, metabolic syndrome, and diabetes.

Senescence exerts a great impact on both biological and functional properties of circulating endothelial progenitor cells (EPCs), especially in cardiovascular diseases where the physiological process of aging is accelerated upon clinical administration of certain drugs such as doxorubicin. EPC impairment contributes to doxorubicin-induced cardiotoxicity. Doxorubicin accelerates EPC aging, although mechanisms underlying this phenomenon remain to be fully clarified. Here we investigated if Nox2 activity is able to modulate the premature senescence induced in vitro by doxorubicin in human EPCs. Results showed that in conditioned media obtained from late EPC cultures, the levels of interleukin-6, isoprostanes and nitric oxide bioavailability were increased and reduced respectively after 3 h of doxorubicin treatment. These derangements returned to physiological levels when cells were co-treated with apocynin or gp91ds-tat (antioxidant and specific Nox2 inhibitors, respectively). Accordingly, Nox2 activity resulted to be activated by doxorubicin. Importantly, we found that Nox2 inhibition reduced doxorubicin-induced EPC senescence, as indicated by a lower percentage of β-gal positive EPCs. In conclusion, Nox2 activity efficiently contributes to the mechanism of oxidative stress-induced increase in premature aging conferred by doxorubicin. The importance of modulation of Nox2 in human EPCs could reveal a useful tool to restore EPC physiological function and properties.

Β-blockers (BB) are a primary treatment for chronic heart disease (CHD), resulting in prognostic and symptomatic benefits. Cardiac cell therapy represents a promising regenerative treatment and, for autologous cell therapy, the patients clinical history may correlate with the biology of resident progenitors and the quality of the final cell product. This study aimed at uncovering correlations between clinical records of biopsy-donor CHD patients undergoing cardiac surgery and the corresponding yield and phenotype of cardiospheres (CSs) and CS-derived cells (CDCs), which are a clinically relevant population for cell therapy, containing progenitors. We describe a statistically significant association between BB therapy and improved CSs yield and CDCs phenotype. We show that BB-CDCs have a reduced fibrotic-like CD90 + subpopulation, with reduced expression of collagen-I and increased expression of cardiac genes, compared to CDCs from non-BB donors. Moreover BB-CDCs had a distinctive microRNA expression profile, consistent with reduced fibrotic features (miR-21, miR-29a/b/c downregulation), and enhanced regenerative potential (miR-1, miR-133, miR-101 upregulation) compared to non-BB. In vitro adrenergic pharmacological treatments confirmed cytoprotective and anti-fibrotic effects of β1-blocker on CDCs. This study shows anti-fibrotic and pro-commitment effects of BB treatment on endogenous cardiac reparative cells, and suggests adjuvant roles of β-blockers in cell therapy applications.

Caloric restriction mimetics (CRMs) are emerging as potential therapeutic agents for the treatment of cardiovascular diseases. CRMs include natural and synthetic compounds able to inhibit protein acetyltransferases, to interfere with acetyl coenzyme A biosynthesis, or to activate (de)acetyltransferase proteins. These modifications mimic the effects of caloric restriction, which is associated with the activation of autophagy. Previous evidence demonstrated the ability of CRMs to ameliorate cardiac function and reduce cardiac hypertrophy and maladaptive remodelling in animal models of ageing, mechanical overload, chronic myocardial ischaemia, and in genetic and metabolic cardiomyopathies. In addition, CRMs were found to reduce acute ischaemia-reperfusion injury. In many cases, these beneficial effects of CRMs appeared to be mediated by autophagy activation. In the present review, we discuss the relevant literature about the role of different CRMs in animal models of cardiac diseases, emphasizing the molecular mechanisms underlying the beneficial effects of these compounds and their potential future clinical application.

The mitochondrial uncoupling protein 2 (UCP2) plays a protective function in the vascular disease of both animal models and humans. UCP2 downregulation upon high-salt feeding favors vascular dysfunction in knock-out mice, and accelerates cerebrovascular and renal damage in the stroke-prone spontaneously hypertensive rat. Overexpression of UCP2 counteracts the negative effects of high-salt feeding in both animal models. We tested in vitro the ability of UCP2 to stimulate autophagy and mitophagy as a mechanism mediating its protective effects upon high-salt exposure in endothelial and renal tubular cells. UCP2 silencing reduced autophagy and mitophagy, whereas the opposite was true upon UCP2 overexpression. High-salt exposure increased level of reactive oxygen species (ROS), UCP2, autophagy and autophagic flux in both endothelial and renal tubular cells. In contrast, high-salt was unable to induce autophagy and autophagic flux in UCP2-silenced cells, concomitantly with excessive ROS accumulation. The addition of an autophagy inducer, Tat-Beclin 1, rescued the viability of UCP2-silenced cells even when exposed to high-salt. In summary, UCP2 mediated the interaction between high-salt-induced oxidative stress and autophagy to preserve viability of both endothelial and renal tubular cells. In the presence of excessive ROS accumulation (achieved upon UCP2 silencing and high-salt exposure of silenced cells) autophagy was turned off. In this condition, an exogenous autophagy inducer rescued the cellular damage induced by excess ROS level. Our data confirm the protective role of UCP2 toward high-salt-induced vascular and renal injury, and they underscore the role of autophagy/mitophagy as a mechanism counteracting the high-salt-induced oxidative stress damage.

Significance: The prevalence of obesity and cardiometabolic phenotypes is alarmingly increasing across the globe and is associated with atherosclerotic vascular complications and high mortality. In spite of multifactorial interventions, vascular residual risk remains high in this patient population, suggesting the need for breakthrough therapies. The mechanisms underpinning obesity-related vascular disease remain elusive and represent an intense area of investigation. Recent Advances: Epigenetic modifications-defined as environmentally induced chemical changes of DNA and histones that do not affect DNA sequence-are emerging as a potent modulator of gene transcription in the vasculature and might significantly contribute to the development of obesity-induced endothelial dysfunction. DNA methylation and histone post-translational modifications cooperate to build complex epigenetic signals, altering transcriptional networks that are implicated in redox homeostasis, mitochondrial function, vascular inflammation, and perivascular fat homeostasis in patients with cardiometabolic disturbances. Critical Issues: Deciphering the epigenetic landscape in the vasculature is extremely challenging due to the complexity of epigenetic signals and their function in regulating transcription. An overview of the most important epigenetic pathways is required to identify potential molecular targets to treat or prevent obesity-related endothelial dysfunction and atherosclerotic disease. This would enable the employment of precision medicine approaches in this setting. Future Directions: Current and future research efforts in this field entail a better definition of the vascular epigenome in obese patients as well as the unveiling of novel, cell-specific chromatin-modifying drugs that are able to erase specific epigenetic signals that are responsible for maladaptive transcriptional alterations and vascular dysfunction in obese patients. Antioxid. Redox Signal. 34, 1165-1199.

Methylation of non-histone proteins is emerging as a central regulatory mechanism in health and disease. The methyltransferase SETD7 has shown to methylate and alter the function of a variety of proteins in vitro; however, its function in the heart is poorly understood. The present study investigates the role of SETD7 in myocardial ischaemic injury.Experiments were performed in neonatal rat ventricular myocytes (NRVMs), SETD7 knockout mice (SETD7-/-) undergoing myocardial ischaemia/reperfusion (I/R) injury, left ventricular (LV) myocardial samples from patients with ischaemic cardiomyopathy (ICM), and peripheral blood mononuclear cells (PBMCs) from patients with ST-elevation MI (STEMI). We show that SETD7 is activated upon energy deprivation in cultured NRVMs and methylates the Hippo pathway effector YAP, leading to its cytosolic retention and impaired transcription of antioxidant genes manganese superoxide dismutase (MnSOD) and catalase (CAT). Such impairment of antioxidant defence was associated with mitochondrial reactive oxygen species (mtROS), organelle swelling, and apoptosis. Selective pharmacological inhibition of SETD7 by (R)-PFI-2 restored YAP nuclear localization, thus preventing mtROS, mitochondrial damage, and apoptosis in NRVMs. In mice, genetic deletion of SETD7 attenuated myocardial I/R injury, mtROS, and LV dysfunction by restoring YAP-dependent transcription of MnSOD and CAT. Moreover, in cardiomyocytes isolated from I/R mice and ICM patients, (R)-PFI-2 prevented mtROS accumulation, while improving Ca2+-activated tension. Finally, SETD7 was up-regulated in PBMCs from STEMI patients and negatively correlated with MnSOD and CAT.We show a methylation-dependent checkpoint regulating oxidative stress during myocardial ischaemia. SETD7 inhibition may represent a valid therapeutic strategy in this setting.

Long noncoding RNAs (lncRNAs) are emerging as critical regulators of heart physiology and disease, although the studies unveiling their modes of action are still limited to few examples. We recently identified pCharme, a chromatin-associated lncRNA whose functional knockout in mice results in defective myogenesis and morphological remodeling of the cardiac muscle. Here, we combined Cap-Analysis of Gene Expression (CAGE), single-cell (sc)RNA sequencing, and whole-mount in situ hybridization analyses to study pCharme cardiac expression. Since the early steps of cardiomyogenesis, we found the lncRNA being specifically restricted to cardiomyocytes, where it assists the formation of specific nuclear condensates containing MATR3, as well as important RNAs for cardiac development. In line with the functional significance of these activities, pCharme ablation in mice results in a delayed maturation of cardiomyocytes, which ultimately leads to morphological alterations of the ventricular myocardium. Since congenital anomalies in myocardium are clinically relevant in humans and predispose patients to major complications, the identification of novel genes controlling cardiac morphology becomes crucial. Our study offers unique insights into a novel lncRNA-mediated regulatory mechanism promoting cardiomyocyte maturation and bears relevance to Charme locus for future theranostic applications.

C2238 atrial natriuretic peptide (ANP) minor allele (substitution of thymidine with cytosine in position 2238) associates with increased risk of cardiovascular events.We investigated the mechanisms underlying the vascular effects of C2238-αANP.In vitro, human umbilical vein endothelial cell were exposed to either wild-type (T2238)- or mutant (C2238)-αANP. Cell survival and apoptosis were tested by Trypan blue, annexin V, and cleaved caspase-3 assays. C2238-αANP significantly reduced human umbilical vein endothelial cell survival and increased apoptosis. In addition, C2238-αANP reduced endothelial tube formation, as assessed by matrigel. C2238-αANP did not differentially modulate natriuretic peptide receptor (NPR)-A/B activity with respect to T2238-αANP, as evaluated by intracellular cGMP levels. In contrast, C2238-αANP, but not T2238-αANP, markedly reduced intracellular cAMP levels in an NPR-C-dependent manner. Accordingly, C2238-αANP showed higher affinity binding to NPR-C, than T2238-αANP. Either NPR-C inhibition by antisense oligonucleotide or NPR-C gene silencing by small interfering RNA rescued survival and tube formation of human umbilical vein endothelial cell exposed to C2238-αANP. Similar data were obtained in human aortic endothelial cell with NPR-C knockdown. NPR-C activation by C2238-αANP inhibited the protein kinase A/Akt1 pathway and increased reactive oxygen species. Adenovirus-mediated Akt1 reactivation rescued the detrimental effects of C2238-αANP. Overall, these data indicate that C2238-αANP affects endothelial cell integrity through NPR-C-dependent inhibition of the cAMP/protein kinase A/Akt1 pathway and increased reactive oxygen species production. Accordingly, C2238-αANP caused impairment of acetylcholine-dependent vasorelaxation ex vivo, which was rescued by NPR-C pharmacological inhibition. Finally, subjects carrying C2238 minor allele showed early endothelial dysfunction, which highlights the clinical relevance of our results.C2238-αANP reduces endothelial cell survival and impairs endothelial function through NPR-C signaling. NPR-C targeting represents a potential strategy to reduce cardiovascular risk in C2238 minor-allele carriers.

In the past several years, it has been demonstrated that the reactive oxygen species (ROS) may act as intracellular signalling molecules to activate or inhibit specific signalling pathways and regulate physiological cellular functions. It is now well-established that ROS regulate autophagy, an intracellular degradation process. However, the signalling mechanisms through which ROS modulate autophagy in a regulated manner have only been minimally clarified. NADPH oxidase (Nox) enzymes are membrane-bound enzymatic complexes responsible for the dedicated generation of ROS. Different isoforms of Nox exist with different functions. Recent studies demonstrated that Nox-derived ROS can promote autophagy, with Nox2 and Nox4 representing the isoforms of Nox implicated thus far. Nox2- and Nox4-dependent autophagy plays an important role in the elimination of pathogens by phagocytes and in the regulation of vascular- and cancer-cell survival. Interestingly, we recently found that Nox is also important for autophagy regulation in cardiomyocytes. We found that Nox4, but not Nox2, promotes the activation of autophagy and survival in cardiomyocytes in response to nutrient deprivation and ischaemia through activation of the PERK (protein kinase RNA-like endoplasmic reticulum kinase) signalling pathway. In the present paper, we discuss the importance of Nox family proteins and ROS in the regulation of autophagy, with a particular focus on the role of Nox4 in the regulation of autophagy in the heart.

UCP2 maps nearby the lod score peak of STR1-stroke QTL in the SHRSP rat strain. We explored the potential contribution of UCP2 to the high-salt diet (JD)-dependent increased stroke susceptibility of SHRSP. Male SHRSP, SHRSR, two reciprocal SHRSR/SHRSP-STR1/QTL stroke congenic lines received JD for 4 weeks to detect brain UCP2 gene/protein modulation as compared with regular diet (RD). Brains were also analyzed for NF-κB protein expression, oxidative stress level and UCP2-targeted microRNAs expression level. Next, based on knowledge that fenofibrate and Brassica Oleracea (BO) stimulate UCP2 expression through PPARα activation, we monitored stroke occurrence in SHRSP receiving JD plus fenofibrate versus vehicle, JD plus BO juice versus BO juice plus PPARα inhibitor. Brain UCP2 expression was markedly reduced by JD in SHRSP and in the (SHRsr.SHRsp-(D1Rat134-Mt1pa)) congenic line, whereas NF-κB expression and oxidative stress level increased. The opposite phenomenon was observed in the SHRSR and in the (SHRsp.SHRsr-(D1Rat134-Mt1pa)) reciprocal congenic line. Interestingly, the UCP2-targeted rno-microRNA-503 was significantly upregulated in SHRSP and decreased in SHRSR upon JD, with consistent changes in the two reciprocal congenic lines. Both fenofibrate and BO significantly decreased brain microRNA-503 level, upregulated UCP2 expression and protected SHRSP from stroke occurrence. In vitro overexpression of microRNA-503 in endothelial cells suppressed UCP2 expression and led to a significant increase of cell mortality with decreased cell viability. Brain UCP2 downregulation is a determinant of increased stroke predisposition in high-salt-fed SHRSP. In this context, UCP2 can be modulated by both pharmacological and nutraceutical agents. The microRNA-503 significantly contributes to mediate brain UCP2 downregulation in JD-fed SHRSP.

ANP (atrial natriuretic peptide), discovered 30 years ago in rat cardiac atria, has been extensively investigated with regard to physiology, pathophysiology, cardiovascular disease therapeutics and molecular genetic aspects. Besides its diuretic, natriuretic and vasorelaxant effects, novel properties of this hormone have been described. Thus anti-hypertrophic, anti-fibrotic, anti-proliferative and anti-inflammatory actions suggest that ANP contributes not only to haemodynamic homoeostasis and adjustments, but has also a role in cardiovascular remodelling. Circulating ANP levels represent a valuable biomarker in cardiovascular diseases. ANP structure is highly conserved among species, indicating a key role in cardiovascular health. Thus an abnormal ANP structure may contribute to an increased risk of disease due to altered functions at either the vascular or cardiac level. Among others, the 2238T&amp;gt;C exon 3 variant has been associated with endothelial cell damage and dysfunction and with an increased risk of acute cardiovascular events, a frameshift mutation within exon 3 has been related to increased risk of atrial fibrillation, and ANP gene variants have been linked to increased risk of hypertension in different ethnic groups. On the other hand, the rs5068 variant, falling within the 3′ UTR and associated with higher circulating ANP levels, has been shown to have a beneficial cardioprotective and metabolic effect. Dissecting out the disease mechanisms dependent on specific ANP molecular variants may reveal information useful in the clinical setting for diagnostic, prognostic and therapeutic purposes. Furthermore, insights from molecular genetic analysis of ANP may well integrate advancing knowledge on the role of ANP as a significant biomarker in patients affected by cardiovascular diseases.

Massive pulmonary embolism (PE) is a severe condition that can potentially lead to death caused by right ventricular (RV) failure and the consequent cardiogenic shock. Despite the fact thrombolysis is often administrated to critical patients to increase pulmonary perfusion and to reduce RV afterload, surgical treatment represents another valid option in case of failure or contraindications to thrombolytic therapy. Correct risk stratification and multidisciplinary proactive teams are critical factors to dramatically decrease the mortality of this global health burden. In fact, the worldwide incidence of PE is 60-70 per 100,000, with a mortality ranging from 1% for small PE to 65% for massive PE. This review provides an overview of the diagnosis and management of this highly lethal pathology, with a focus on the surgical approaches at the state of the art.

Intensive physical exercise may cause increase oxidative stress and muscular injury in elite football athletes. The aim of this study was to exploit the effect of cocoa polyphenols on oxidative stress and muscular injuries induced by intensive physical exercise in elite football players. Oxidant/antioxidant status and markers of muscle damage were evaluated in 24 elite football players and 15 controls. Furthermore, the 24 elite football players were randomly assigned to either a dark chocolate (&gt;85% cocoa) intake (<mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" id="M1"><mml:mi>n</mml:mi><mml:mo>=</mml:mo><mml:mn>12</mml:mn></mml:math>) or a control group (<mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" id="M2"><mml:mi>n</mml:mi><mml:mo>=</mml:mo><mml:mn>12</mml:mn></mml:math>) for 30 days in a randomized controlled trial. Oxidative stress, antioxidant status, and muscle damage were assessed at baseline and after 30 days of chocolate intake. Compared to controls, elite football players showed lower antioxidant power and higher oxidative stress paralleled by an increase in muscle damage markers. After 30 days of dark chocolate intake, an increased antioxidant power was found in elite athletes assuming dark chocolate. Moreover, a significant reduction in muscle damage markers (CK and LDH, <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" id="M3"><mml:mi>p</mml:mi><mml:mo>&lt;</mml:mo><mml:mn>0.001</mml:mn></mml:math>) was observed. In the control group, no changes were observed with the exception of an increase of sNox2-dp, H 2 O 2 , and myoglobin. A simple linear regression analysis showed that sNox2-dp was associated with a significant increase in muscle damage biomarker release (<mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" id="M4"><mml:mi>p</mml:mi><mml:mo>=</mml:mo><mml:mn>0.001</mml:mn></mml:math>). An in vitro study also confirmed that polyphenol extracts significantly decreased oxidative stress in murine myoblast cell line C2C12-derived. These results indicate that polyphenol-rich nutrient supplementation by means of dark chocolate positively modulates redox status and reduced exercise-induced muscular injury biomarkers in elite football athletes. This trial is registered with NCT03288623 .

Oxidative states exert a significant influence on a wide range of biological and molecular processes and functions. When their balance is shifted towards enhanced amounts of free radicals, pathological phenomena can occur, as the generation of reactive oxygen species (ROS) in tissue microenvironment or in the systemic circulation can be detrimental. Epidemic chronic diseases of western societies, such as cardiovascular disease, obesity, and diabetes correlate with the imbalance of redox homeostasis. Current advances in our understanding of epigenetics have revealed a parallel scenario showing the influence of oxidative stress as a major regulator of epigenetic gene regulation via modification of DNA methylation, histones, and microRNAs. This has provided both the biological link and a potential molecular explanation between oxidative stress and cardiovascular/metabolic phenomena. Accordingly, in this review, we will provide current insights on the physiological and pathological impact of changes in oxidative states on cardiovascular disorders, by specifically focusing on the influence of epigenetic regulation. A special emphasis will highlight the effect on epigenetic regulation of human's current life habits, external and environmental factors, including food intake, tobacco, air pollution, and antioxidant-based approaches. Additionally, the strategy to quantify oxidative states in humans in order to determine which biological marker could best match a subject's profile will be discussed.

Background and aim Impaired fasting glucose (IFG) is associated with an increased risk of cardiovascular disease but the underlying mechanisms are still unclear. Aim of the study was to investigate the interplay between platelet activation, lipopolysaccharides (LPS) and markers of oxidative stress in patients with IFG and control subjects. Methods and results We performed a cross-sectional study including 35 patients with IFG and 35 control subjects who were well comparable for age, sex, body mass index and smoking history. Serum levels of LPS, zonulin (a marker of gut permeability), oxidized LDL and plasma levels of soluble P-selectin, were measured. Patients with IFG had significantly higher levels of sP-selectin, LPS, zonulin and oxLDL compared to control subjects. The IFG status (beta coefficient: 0.518, p < 0.001), higher LPS (beta coefficient: 0.352, p = 0.001) and female sex (beta coefficient: 0.179, p = 0.042) were independently associated with higher sP-selectin; in addition, oxLDL was positively associated with sP-selectin (r = 0.530, p < 0.001) and LPS (r = 0.529, p = 0.001). In IFG patients, we found a significant association between LPS and zonulin (r = 0.521, p = 0.001); this association was confirmed at multivariable analysis (beta coefficient: 0.512, p = 0.007). Conclusion Our study provides evidence that patients with IFG have increased platelet activation, and suggests LPS as a potential trigger for in vivo platelet activation in this patient population.

Patients with diabetes are more prone to developing heart failure in the presence of high blood pressure than those without diabetes. Yes-associated protein (YAP), a key effector of the Hippo signaling pathway, is persistently activated in diabetic hearts, and YAP plays an essential role in mediating the exacerbation of heart failure in response to pressure overload in the hearts of mice fed a high-fat diet. YAP induced dedifferentiation of cardiomyocytes through activation of transcriptional enhancer factor 1 (TEAD1), a transcription factor. Thus, YAP and TEAD1 are promising therapeutic targets for diabetic patients with high blood pressure to prevent the development of heart failure.

The impact of seasonal changes on the incidence of acute myocardial infarction has been incompletely appraised, especially in the modern era of primary percutaneous coronary intervention (PPCI). We aimed to appraise the overall and season-specific impact of climate changes on the daily rate of PCCI.Details on PPCI and climate changes were retrospectively collected in three high-volume Italian institutions with different geographical features. The association between rate of PPCI and temperature, atmospheric pressure (ATM), humidity and rainfall was appraised with Poisson models, with overall analyses and according to season of the year.Details on 6880 days with a total of 4132 PPCI were collected. Overall adjusted analysis showed that higher minimum atmospheric pressure 3 days before PPCI were associated with lower risk (regression coefficient = 0.999 [95% confidence interval 0.998-1.000], p = 0.030). Focusing on season, in Winter PPCI rates were increased by lower same day mean temperature (0.973 [0.956-0.990], p = 0.002) and lower rainfall (0.980 [0.960-1.000], p = 0.049). Conversely, in Spring greater changes in atmospheric pressure 3 days before PPCI were associated with increased risk (1.023 [1.002-1.045], p = 0.032), with similar effects in Summer for minimum temperature on the same day (1.022 [1.001-1.044], p = 0.040).Climate has a significant impact on the risk of PPCI in the current era, with a complex interplay according to season. Higher risk risk is expected with lower minimum atmospheric pressure in the preceding days, lower rainfall in Winter, greater changes in atmospheric pressure in Spring, and higher temperatures in Summer. These findings have important implications for prevention strategies.

Bicuspid aortic valve (BAV) is a common congenital heart malformation frequently associated with the development of aortic valve diseases and severe aortopathy, such as aortic dilatation, aneurysm and dissection. To date, different genetic loci have been identified in syndromic and non- syndromic forms of BAV. Among these, genes involved in the regulation of extracellular matrix remodelling, epithelial to mesenchymal transition and nitric oxide metabolism appear to be the main contributors to BAV pathogenesis. However, no- single gene model explains BAV inheritance, suggesting that more factors are simultaneously involved. In this regard, characteristic epigenetic and immunological profiles have been documented to contradistinguish BAV individuals. In this review, we provide a comprehensive overview addressing molecular mechanisms involved in BAV development and progression.

Environmental pollution and weather changes unfavorably impact on cardiovascular disease. However, limited research has focused on ST-elevation myocardial infarction (STEMI), the most severe yet distinctive form of acute coronary syndrome.We appraised the impact of environmental and weather changes on the incidence of STEMI, analysing the bivariate and multivariable association between several environmental and atmospheric parameters and the daily incidence of STEMI in two large Italian urban areas. Specifically, we appraised: carbon monoxide (CO), nitrogen dioxide (NO2), nitric oxide (NOX), ozone, particulate matter smaller than 10 μm (PM10) and than 2.5 μm (PM2.5), temperature, atmospheric pressure, humidity and rainfall. A total of 4285 days at risk were appraised, with 3473 cases of STEMI. Specifically, no STEMI occurred in 1920 (44.8%) days, whereas one or more occurred in the remaining 2365 (55.2%) days. Multilevel modelling identified several pollution and weather predictors of STEMI. In particular, concentrations of CO (p=0.024), NOX (p=0.039), ozone (p=0.003), PM10 (p=0.033) and PM2.5 (p=0.042) predicted STEMI as early as three days before the event, as well as subsequently, and NO predicted STEMI one day before (p = 0.010), as well as on the same day. A similar predictive role was evident for temperature and atmospheric pressure (all p < 0.05).The risk of STEMI is strongly associated with pollution and weather features. While causation cannot yet be proven, environmental and weather changes could be exploited to predict STEMI risk in the following days.

Background and Purpose Oxidative stress and insufficient autophagy activity are associated with inflammatory processes and are common features of many cardiovascular diseases (CVDs). We investigated if a combination of natural activators of autophagy could modulate oxidative stress, platelet aggregation and endothelial cell survival and function in response to stress. Experimental Approach Ex vivo platelet aggregation and activation, H 2 O 2 production and autophagy were measured in platelets of subjects at high cardiovascular risk, including smokers, patients with metabolic syndrome (MetS) and patients with atrial fibrillation (AF). In vitro, the effects of a mixture of natural pro‐autophagy molecules and antioxidants on platelets and human umbilical vein endothelial cells (HUVECs) were evaluated. Key Result Autophagy appeared to be inhibited, whereas aggregation was increased in platelets from AF and MetS patients and in smokers, as compared with healthy subjects. Treatment of platelets isolated from these patients with a mixture composed of trehalose, spermidine, catechin and epicatechin (Mix1) or with a mixture composed of trehalose, spermidine and nicotinamide (Mix2), significantly reduced platelet activation and oxidative stress, and increased autophagy, compared with the effect of each compound alone. Similarly, treatment of HUVECs with a combination of these compounds exhibited beneficial effects and increased endothelial cell survival, nitric oxide bioavailability and angiogenesis in response to stress in a potentiated manner. Conclusion and Implications A combination of natural activators of autophagy could inhibit platelet activity and oxidative stress and improve endothelial cell survival and function in a potentiated manner representing a useful strategy to reduce the effect of risk factors on CVD occurrence. LINKED ARTICLES This article is part of a themed issue on Cellular metabolism and diseases. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v178.10/issuetoc

Patients with Coronavirus-associated disease-2019 (COVID-19) display alterations of the hemostatic system and the presence of a prothrombotic status frequently leading to vascular complications. However, the impact of COVID-19 on platelet activity, aggregation and agglutination still needs to be clarified. We measured total levels of von Willebrand factor (vWF) and vWF binding to the platelet glycoprotein (Gp) complex (GPIb-IX-V), in a cohort of COVID-19 patients admitted to the intensive care unit of our Institution. Moreover, we evaluated platelet aggregation in response to agonists (ADP, collagen, arachidonic acid) and platelet agglutination in response to ristocetin. We found that levels of vWF antigen and the active form of vWF binding to platelets (vWF:RCo), were markedly increased in these patients. These results were associated with higher agglutination rates induced by ristocetin, thereby indirectly indicating an increased capability of vWF to bind to platelets. Conversely, we found that platelet aggregation in response to both ADP and collagen was lower in COVID-19 patients compared to healthy volunteers. This study shows that COVID-19 is associated with increased vWF-induced platelet agglutination but reduced platelet responsivity to aggregation stimuli. Our findings have translational relevance since platelet adhesion to vWF may represent a marker to predict possible complications and better delineate therapeutic strategies in COVID-19 patients.

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.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.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.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.

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.

To evaluate the effects of treatments based on angiotensin II receptor blockers (ARBs) on the risk of myocardial infarction (MI), cardiovascular and all-cause death, as compared with conventional treatment or placebo.We performed a meta-analysis of all available major international, randomized clinical trials (20 trials, n = 108 909 patients, mean age 66.5 +/- 4.1 years), published by 31 August 2008, comparing ARBs with other drugs or conventional therapies (placebo) and reporting MI incidence.During a mean follow-up of 3.3 +/- 1.1 years, a total of 2374/53 208 and 2354/53 153 cases of MI were recorded in ARB-based groups and in comparator arms, respectively [odds ratio (OR) 95% confidence interval (CI) 1.008 (0.950-1.069)]. Risks of MI were not different when tested in different clinical conditions, including hypertension, high cardiovascular risk, stroke, coronary disease, renal disease and heart failure. No significant differences in the risk of MI between treatment with ARBs versus placebo [OR 95% CI 0.944 (0.841-1.060)], beta-blockers and diuretics [OR 95% CI 0.970 (0.804-1.170)], calcium channel blockers [OR 95% CI 1.112 (0.971-1.272)], or angiotensin-converting enzyme (ACE) inhibitors [OR 95% CI 1.008 (0.926-1.099)] were observed. Analysis of trials comparing combination therapy based on ARBs plus ACE inhibitors versus active treatments or placebo showed equivalent MI risk [OR 95% CI 0.996 (0.896-1.107)].The present meta-analysis indicates that the risk of MI is comparable with use of ARBs and other antihypertensive drugs in a wide range of clinical conditions.

Metabolic syndrome (MetS) and type 2 diabetes (T2DM) have been associated with an impairment of left (LV) and right ventricular (RV) function as well as an increased risk of heart failure (HF). However, it remains unclear whether these clinical entities or their associations promote a similar derangement of biventricular function. Overall, 345 patients without overt cardiovascular disease consecutively underwent routine blood chemistry including high-sensitivity C reactive protein (hs-CRP) and echocardiographical examination with conventional and tissue Doppler imaging (TDI) of both ventricles. According to the ATP III criteria and fasting glucose levels, the study population was stratified into four groups: (1) healthy controls (n=120); (2) MetS without T2DM (n=84); (3) T2DM without MetS (n=49); and (4) MetS+T2DM (n=92). The Myocardial performance index (MPI) of the RV and LV was obtained with a multi-segmental approach using TDI. Patients with MetS and T2DM exhibited a similar impairment of biventricular function compared with healthy controls, whereas a further decline was observed in patients having both MetS and T2DM. In addition to MetS markers, hs-CRP exhibited the strongest association with the MPI of both ventricles. Regression analyses indicated that individual MetS markers were inferior to MetS in identifying subtle cardiac dysfunction. Independent associations of MetS and T2DM with biventricular dysfunction were comparable, and the coexistence of MetS and T2DM exhibited the highest risk for biventricular dysfunction. Our findings emphasize the importance of MetS as an equivalent of T2DM and support a synergic effect of these clinical conditions on cardiac organ damage requiring more aggressive therapeutic strategies to prevent HF.

Ndufc2, a subunit of the NADH: ubiquinone oxidoreductase, plays a key role in the assembly and activity of complex I within the mitochondrial OXPHOS chain. Its deficiency has been shown to be involved in diabetes, cancer and stroke. To improve our knowledge on the mechanisms underlying the increased disease risk due to Ndufc2 reduction, we performed the present in vitro study aimed at the fine characterization of the derangements in mitochondrial structure and function consequent to Ndufc2 deficiency. We found that both fibroblasts obtained from skin of heterozygous Ndufc2 knock-out rat model showed marked mitochondrial dysfunction and PBMC obtained from subjects homozygous for the TT genotype of the rs11237379/NDUFC2 variant, previously shown to associate with reduced gene expression, demonstrated increased generation of reactive oxygen species and mitochondrial damage. The latter was associated with increased oxidative stress and significant ultrastructural impairment of mitochondrial morphology with a loss of internal cristae. In both models the exposure to stress stimuli, such as high-NaCl concentration or LPS, exacerbated the mitochondrial damage and dysfunction. Resveratrol significantly counteracted the ROS generation. These findings provide additional insights on the role of an altered pattern of mitochondrial structure-function as a cause of human diseases. In particular, they contribute to underscore a potential genetic risk factor for cardiovascular diseases, including stroke.

Accumulating lines of evidence suggest that reactive oxygen species (ROS) may act as intracellular signaling molecules under cellular stress conditions, activating several molecular pathways. Autophagy, the intracellular mechanism by which cells digest and recycle unfolded proteins and dysfunctional organelles, is emerging as a major target of ROS and NADPH oxidase (Nox) enzymes, the major generators of ROS. While autophagy represents an important self-defense mechanism in promoting cell survival, it may be maladaptive in some conditions. In particular, in the cardiovascular system, moderate activation of autophagy has been shown to be protective, while excessive or insufficient activation of autophagy may be deleterious. Thus, modulating ROS-dependent autophagy may represent a novel strategy to keep autophagy within the therapeutic range. Among the Nox isoforms, Nox4 in particular plays a pivotal role in autophagy regulation. This appears to be due to its intracellular localization and its ability to produce hydrogen peroxide, a stable signaling molecule. In this chapter we review the studies relating to the functional role of Nox4 in autophagy, with particular emphasis on the heart and cardiovascular system.

HomeCirculation ResearchVol. 123, No. 8Novel Beneficial Cardiovascular Effects of Natural Activators of Autophagy Free AccessArticle CommentaryPDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toFree AccessArticle CommentaryPDF/EPUBNovel Beneficial Cardiovascular Effects of Natural Activators of Autophagy Giacomo Frati, Carmine Vecchione and Sebastiano Sciarretta Giacomo FratiGiacomo Frati From the Department of Medico-Surgical Sciences and Biotechnologies, Sapienza University of Rome, Latina, Italy (G.F., S.S.) Department of AngioCardioNeurology, IRCCS Neuromed, Pozzilli (IS), Italy (G.F., C.V., S.S.) , Carmine VecchioneCarmine Vecchione Department of AngioCardioNeurology, IRCCS Neuromed, Pozzilli (IS), Italy (G.F., C.V., S.S.) University of Salerno, Medicine and Surgery, Baronissi, Italy (C.V.). and Sebastiano SciarrettaSebastiano Sciarretta Correspondence to Sebastiano Sciarretta, MD, PhD, Department of Medico-Surgical Sciences and Biotechnologies, Sapienza University of Rome, Corso della Repubblica 74, 04100 Latina, Italy. Email E-mail Address: [email protected] From the Department of Medico-Surgical Sciences and Biotechnologies, Sapienza University of Rome, Latina, Italy (G.F., S.S.) Department of AngioCardioNeurology, IRCCS Neuromed, Pozzilli (IS), Italy (G.F., C.V., S.S.) Originally published27 Sep 2018https://doi.org/10.1161/CIRCRESAHA.118.313530Circulation Research. 2018;123:947–949Novel natural activators of autophagy are able to exert extraordinary beneficial effects in the cardiovascular system. This evidence suggests that these compounds may be potentially useful for the treatment of cardiovascular diseases.Autophagy is an intracellular process that is in charge for the degradation of senescent or dysfunctional cellular components through their sequestration by vesicles called autophagosomes and their subsequent degradation in lysosomes. The primary molecules derived from this catabolic process are finally recycled and reused for other cellular purposes.1 Autophagy is a paramount process for the regulation of cellular homeostasis particularly in response to stress. Activation of autophagy limits myocardial damage induced by ischemia, chronic myocardial infarction, mechanical stress, and heart failure.1 Autophagy also attenuates vascular damage and lessens atherosclerosis development.2,3In the past few years, several natural compounds have gained momentum because of their ability to exert beneficial cardiovascular effects and to activate autophagy.Trehalose is a nonreducing sugar made of 2 molecules of glucose joined by means of an α-1,1 glycosidic linkage.4 It is a natural disaccharide synthetized by diverse life beings, such as yeasts, tardigrades, worms, insects, and vegetables, particularly during stress. On the contrary, trehalose cannot be synthetized by mammals, including humans, because of the lack of biosynthesis genes. The scientific attention to this sugar raised over the years because trehalose synthesis increases life span in lower organisms and protects them against injury caused by desiccation, extreme temperatures, reactive oxygen species, and misfolded protein accumulation.4 Exogenous administration of trehalose was also shown to protect mammalian cells in response to stress.4,5 Interestingly, Sarkar et al5 demonstrated that trehalose has a great ability to increase autophagy independently of mTOR (mammalian target of rapamycin) signaling.Trehalose is also potentially able to treat human disease.4 Oral administration of this disaccharide is able to dramatically reduce the development and progression of neurodegenerative disorders, hepatic steatosis, kidney injury, insulin resistance, and pancreatitis, mostly through the stimulation of autophagy. Ingested trehalose is metabolized by intestinal trehalase enzyme, which breaks down the disaccharide into 2 glucose molecules. However, a certain amount of trehalose is still able to pass the gut barrier and to accumulate in peripheral organs after oral intake,6 thereby exerting direct beneficial effects.Trehalose is also able to exert beneficial effects in the cardiovascular system. Trehalose reduced cardiomyocyte death in response to energy stress in cardiomyocytes in vitro.7 Oral administration of trehalose significantly reduced cardiac remodeling, dysfunction, and heart failure in a mouse model of chronic myocardial infarction through the activation of autophagy.8 In fact, trehalose administration significantly activated autophagy and mitophagy in cardiomyocytes, and its beneficial cardiac effects were blunted in Beclin-1+/− mice, which display genetic inhibition of autophagy. Interestingly, Sergin et al3 recently showed that trehalose administration is also able to reduce atherosclerosis development in ApoE−/− (apolipoprotein E) mice fed with high-fat diet. Trehalose administration activated autophagy in macrophages of these mice thereby reducing cytokine production and vascular inflammation. These beneficial effects were abrogated by macrophage-specific conditional deletion of Atg5 gene, indicating that activation of autophagy in these cells is required for trehalose antiatherosclerotic effects.3 Trehalose seems to exert beneficial cardiovascular effects also in human subjects. A recent clinical trial demonstrated that oral administration of this disaccharide reduced arterial aging in healthy middle-aged subjects.9 Mechanistically, trehalose seems to stimulate autophagy through the activation of TFEB (transcription factor EB). Trehalose activated TFEB in cardiomyocytes, and knockdown of this transcription factor attenuated the proautophagic effects of this disaccharide.8 Similarly, the antiatherosclerotic effects of trehalose were associated with TFEB activation.3 Future studies are required to evaluate how trehalose promotes TFEB nuclear localization and activation.Another natural activator of autophagy is spermidine. Spermidine is a polyamine usually found in mammals, including human subjects, in both circulating blood and tissues.10 It is found in different kinds of food, particularly wheat germ, natto, soy beans, and mushrooms.10 However, spermidine content in mammals is also determined by its synthesis rate and catabolism.10Spermidine regulates cellular homeostasis and response to stress, by exerting antioxidant and anti-inflammatory functions and by regulating DNA stability, cellular growth, and survival. In addition, spermidine promotes protein deacetylation, thereby controlling multiple cellular processes. Spermidine is also a potent activator of autophagy, mostly through the inhibition of the histone acetyltransferase P300, although it is also able to activate AMPK (AMP-activated protein kinase) and inhibit mTORC1.10Endogenous spermidine levels decrease during aging, and spermidine treatment extends life span both in lower organisms and in mammals through the activation of autophagy. Epidemiological analyses also showed that the amount of spermidine intake with diet is directly correlated with longevity in human subjects.11 This evidence further corroborates previous data demonstrating that autophagy activation exerts antiaging effects and extends life span.1 Spermidine treatment also promotes neuroprotective functions, inhibits inflammation, reduces the risk of cancer,10 and counteracts insulin resistance development.10 Spermidine also exerts extraordinary cardiovascular beneficial effects. Its oral administration was shown to reduce cardiac senescence in mice, by ameliorating diastolic function and reducing cardiac hypertrophy.11 Spermidine also reduced hypertension-induced cardiac dysfunction and heart failure.11 Spermidine treatment increased autophagy and mitophagy in cardiomyocytes, and the beneficial effects of this natural polyamine were attenuated by autophagy inhibition through cardiomyocyte-restricted Atg5 gene deletion.11 Spermidine also attenuated atherosclerosis formation in ApoE−/− mice in an autophagy-dependent manner12 and improved endothelial function in aged mice.10 It will be important to test in the future whether the effects of spermidine on endothelial function are mediated by autophagy activation.Finally, polyphenols are also able to activate autophagy. Polyphenols are natural compounds contained in several types of food and beverages, such as red wine, green tea, fruits, oils, vegetables, cocoa, and spices, which are advocated to mediate the beneficial health effects associated with the consumption of these aliments.13 Polyphenols such as resveratrol and curcumin exerted beneficial cardiovascular effects in experimental animal models of endothelial dysfunction, atherosclerosis, and cardiac injury,13 although these results were not always translated to human subjects probably because a mixture of different compounds is required for obtaining beneficial effects rather than the administration of a single molecule. Aside from their antioxidant properties observed in vitro, it is now known that polyphenols may also exert beneficial effects through the activation of autophagy. Previous work from Kroemer's laboratory showed that polyphenols contained in red wine are able to stimulate autophagy, likely through protein deacetylation.14 Future studies are needed to understand the role of autophagy activation in beneficial cardiovascular effects of these compounds.Overall, these data strongly suggest that these natural compounds may be used as dietary supplements for the treatment of cardiovascular diseases in combination with standard pharmacological therapy (Figure). It will be important to better dissect the molecular mechanisms through which these molecules regulate autophagy, because it is likely that they control this process through different mechanisms. The elucidation of this issue is particularly important to understand the existence of potential synergistic effects of these compounds. In this regard, future studies are encouraged to test whether a combination of different autophagy activators confers additive cardiovascular protection compared with the effects of single molecules.Download figureDownload PowerPointFigure. Novel natural activators of autophagy. Trehalose, spermidine, and polyphenols exert beneficial cardiovascular effects through the activation of autophagy.It will also be important to test the effects of these natural compounds in clinical trials including patients with cardiovascular diseases. In fact, the exceptional advantage of these natural molecules is the absence or paucity of adverse side effects. Of note, recent work suggested that trehalose may favor the proliferation of specific epidemic subtypes of Clostridium difficile15 in rodents, creating a note of caution on the regular use of this sugar. However, there is no evidence in the literature indicating that trehalose intake favors a C. difficile infection in human subjects. Therefore, future studies are also encouraged to address this important issue.Sources of FundingThis work is partially supported by a grant from the Italian Ministry of Health to Dr Sciarretta (GR-2013-02355401).DisclosuresNone.FootnotesThe opinions expressed in this article are not necessarily those of the editors or of the American Heart Association.Correspondence to Sebastiano Sciarretta, MD, PhD, Department of Medico-Surgical Sciences and Biotechnologies, Sapienza University of Rome, Corso della Repubblica 74, 04100 Latina, Italy. Email sebastiano.[email protected]itReferences1. Sciarretta S, Maejima Y, Zablocki D, Sadoshima J. The role of autophagy in the heart.Annu Rev Physiol. 2018; 80:1–26. doi: 10.1146/annurev-physiol-021317-121427CrossrefMedlineGoogle Scholar2. Pankratz F, Hohnloser C, Bemtgen X, et al. MicroRNA-100 suppresses chronic vascular inflammation by stimulation of endothelial autophagy.Circ Res. 2018; 122:417–432. doi: 10.1161/CIRCRESAHA.117.311428LinkGoogle Scholar3. Sergin I, Evans TD, Zhang X, et al. Exploiting macrophage autophagy-lysosomal biogenesis as a therapy for atherosclerosis.Nat Commun. 2017; 8:15750. doi: 10.1038/ncomms15750CrossrefMedlineGoogle Scholar4. Hosseinpour-Moghaddam K, Caraglia M, Sahebkar A. Autophagy induction by trehalose: molecular mechanisms and therapeutic impacts.J Cell Physiol. 2018; 233:6524–6543. doi: 10.1002/jcp.26583CrossrefMedlineGoogle Scholar5. Sarkar S, Davies JE, Huang Z, Tunnacliffe A, Rubinsztein DC. Trehalose, a novel mTOR-independent autophagy enhancer, accelerates the clearance of mutant huntingtin and alpha-synuclein.J Biol Chem. 2007; 282:5641–5652. doi: 10.1074/jbc.M609532200CrossrefMedlineGoogle Scholar6. Tanaka M, Machida Y, Niu S, Ikeda T, Jana NR, Doi H, Kurosawa M, Nekooki M, Nukina N. 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Versaci F, Valenti V, Forte M, Cammisotto V, Nocella C, Bartimoccia S, Schirone L, Schiavon S, Vecchio D, D'Ambrosio L, Spinosa G, D'Amico A, Chimenti I, Violi F, Frati G, Pignatelli P, Sciarretta S, Pastori D and Carnevale R (2022) Aging-Related Decline of Autophagy in Patients with Atrial Fibrillation—A Post Hoc Analysis of the ATHERO-AF Study, Antioxidants, 10.3390/antiox11040698, 11:4, (698) Abudureyimu M, Yu W, Cao R, Zhang Y, Liu H and Zheng H (2020) Berberine Promotes Cardiac Function by Upregulating PINK1/Parkin-Mediated Mitophagy in Heart Failure, Frontiers in Physiology, 10.3389/fphys.2020.565751, 11 Ameri P, Schiattarella G, Crotti L, Torchio M, Bertero E, Rodolico D, Forte M, Di Mauro V, Paolillo R, Chimenti C, Torella D, Catalucci D, Sciarretta S, Basso C, Indolfi C and Perrino C (2020) Novel Basic Science Insights to Improve the Management of Heart Failure: Review of the Working Group on Cellular and Molecular Biology of the Heart of the Italian Society of Cardiology, International Journal of Molecular Sciences, 10.3390/ijms21041192, 21:4, (1192) Schirone L, Forte M, D'Ambrosio L, Valenti V, Vecchio D, Schiavon S, Spinosa G, Sarto G, Petrozza V, Frati G and Sciarretta S (2022) An Overview of the Molecular Mechanisms Associated with Myocardial Ischemic Injury: State of the Art and Translational Perspectives, Cells, 10.3390/cells11071165, 11:7, (1165) Azam T, Zhang H, Zhou F and Wang X (2022) Recent Advances on Drug Development and Emerging Therapeutic Agents Through Targeting Cellular Homeostasis for Ageing and Cardiovascular Disease, Frontiers in Aging, 10.3389/fragi.2022.888190, 3 Schirone L, D'Ambrosio L, Forte M, Genovese R, Schiavon S, Spinosa G, Iacovone G, Valenti V, Frati G and Sciarretta S (2022) Mitochondria and Doxorubicin-Induced Cardiomyopathy: A Complex Interplay, Cells, 10.3390/cells11132000, 11:13, (2000) September 28, 2018Vol 123, Issue 8 Advertisement Article InformationMetrics © 2018 American Heart Association, Inc.https://doi.org/10.1161/CIRCRESAHA.118.313530PMID: 30355035 Originally publishedSeptember 27, 2018 Keywordsautophagylysosomescardiovascular diseasepharmacologyheart failurePDF download Advertisement SubjectsBasic Science ResearchPathophysiologyPharmacologyTranslational Studies