Atrial natriuretic peptide (ANP) modulates stress-induced autophagy in endothelial cells.

Atrial natriuretic peptide (ANP), a cardiac hormone involved in the regulation of water/sodium balance and blood pressure, is also secreted by endothelial cells, where it exerts protective effects in response to stress. Autophagy is an intracellular self-renewal process involved in the degradation of dysfunctional cytoplasmic elements. ANP was recently reported to act as an extracellular regulator of cardiac autophagy. However, its role in the regulation of endothelial autophagy has never been investigated. Here, we tested the effects of ANP in the regulation of autophagy in human umbilical vein endothelial cells (HUVECs). We found that ANP rapidly increases autophagy and autophagic flux at physiological concentrations through its predominant pathway, mediated by natriuretic peptide receptor type A (NPR-A) and protein kinase G (PKG). We further observed that ANP is rapidly secreted by HUVEC under stress conditions, where it mediates stress-induced autophagy through autocrine and paracrine mechanisms. Finally, we found that the protective effects of ANP in response to high-salt loading or tumor necrosis factor (TNF)-α are blunted by concomitant inhibition of autophagy. Overall, our results suggest that ANP acts as an endogenous autophagy activator in endothelial cells. The autophagy mechanism mediates the protective endothelial effects exerted by ANP.

MST1 mediates doxorubicin-induced cardiomyopathy by SIRT3 downregulation.

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

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

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

Role of Dickkopf-3 in Blood Pressure Regulation in Mice and Hypertensive Rats.

BACKGROUND: Dkk3 (Dickkopf-3) is a secreted glycoprotein known for its proapoptotic and angiogenic activity. The role of Dkk3 in cardiovascular homeostasis is largely unknown. Remarkably, the Dkk3 gene maps within a chromosome segment linked to the hypertensive phenotype in spontaneously hypertensive rats (SHR). METHODS: We used Dkk3-/- mice or stroke-resistant (sr) and stroke-prone (sp) SHR to examine the role of Dkk3 in the central and peripheral regulation of blood pressure (BP). We used lentiviral expression vector to rescue Dkk3 in knockout mice or to induce Dkk3 overexpression or silencing in SHR. RESULTS: Genetic deletion of Dkk3 in mice enhanced BP and impaired endothelium-dependent acetylcholine-induced relaxation of resistance arteries. These alterations were rescued by restoring Dkk3 expression either in the periphery or in the central nervous system (CNS). Dkk3 was required for the constitutive expression of VEGF (vascular endothelium growth factor), and the action of Dkk3 on BP and endothelium-dependent vasorelaxation was mediated by VEGF-stimulated phosphatidylinositol-3-kinase pathway, leading to eNOS (endothelial NO synthase) activation both in resistance arteries and the CNS. The regulatory function of Dkk3 on BP was confirmed in SHR stroke-resistant and SHR stroke-prone in which was blunted in both resistance arteries and brainstem. In SHR stroke-resistant, lentiviral expression vector-induced Dkk3 expression in the CNS largely reduced BP, whereas Dkk3 knock-down further enhanced BP. In SHR stroke-prone challenged with a hypersodic diet, lentiviral expression vector-induced Dkk3 expression in the CNS displayed a substantial antihypertensive effect and delayed the occurrence of stroke. CONCLUSIONS: These findings demonstrate that Dkk3 acts as peripheral and central regulator of BP by promoting VEGF expression and activating a VEGF/Akt (protein kinase B)/eNOS hypotensive axis.

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

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

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

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

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

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

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

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

Impact of a NDUFC2 Variant on the Occurrence of Acute Coronary Syndromes.

Background: Among several potential mechanisms, mitochondrial dysfunction has been proposed to be involved in the pathogenesis of coronary artery disease (CAD). A mitochondrial complex I deficiency severely impairs cardiovascular health and contributes to CAD development. Previous evidence highlighted a key role of NDUFC2, a subunit of complex I, deficiency in the increased occurrence of renal and cerebrovascular damage in an animal model of hypertension, and of juvenile ischemic stroke occurrence in humans. Furthermore, a significant decrease of NDUFC2 mRNA was detected in peripheral blood mononuclear cells from patients experiencing acute coronary syndrome (ACS). The T allele at NDUFC2/rs23117379 variant is known to associate with reduced gene expression and mitochondrial dysfunction. Objective: In the present study we tested the impact of the T/C NDUFC2/rs23117379 variant on occurrence of ACS in a prospective cohort of CAD patients (n = 260). Results: Hypertension, smoking habit, diabetes and hypercholesterolemia were present in a large proportion of patients. Non-ST-elevation myocardial infarction (NSTEMI) represented the most frequent type of ACS (44%, n = 115), followed by ST-elevation myocardial infarction (STEMI) (34%, n = 88) and unstable angina (22%, n = 57). The alleles/genotypes distribution for T/C at NDUFC2/rs23117379 revealed that the TT genotype was associated with a trend toward the development of ACS at an earlier age (TT 61 ± 12, CT 65 ± 12 and CC 66 ± 11 years; p = 0.051 after adjustment for gender, hypertension, smoking habit, diabetes and hypercholesterolemia) and with a significant predictive role for ACS recurrence (hazard ratio [HR]1.671; 95% confidence interval [CI], 1.138-2.472; p = 0.009). Conclusions: Our findings are consistent with a deleterious effect of NDUFC2 deficiency on acute coronary events predisposition and further support a role of the NDUFC2/rs23117379 variant as a genetic cardiovascular risk factor.

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

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

The prognostic role of the echocardiographic tricuspid annular plane systolic excursion/systolic pulmonary arterial pressure (TAPSE/sPAP) ratio and its relationship with NT-proANP plasma level in systemic sclerosis.

Background: The tricuspid annular plane systolic excursion/systolic pulmonary arterial pressure (TAPSE/sPAP) ratio is an echocardiographic estimation of the right ventricle to pulmonary artery (RV/PA) coupling, with a validated prognostic role in different clinical settings. Systemic sclerosis (SSc) patients without evident cardiovascular involvement frequently display subtle RV impairment. The amino-terminal atrial natriuretic peptide (NT-proANP) plasma level relates to SSc disease progression and mortality. We aimed to assess the prognostic value of the TAPSE/sPAP ratio and its relationship with NT-proANP plasma level in SSc patients without overt cardiovascular involvement. Methods: We retrospectively analysed 70 SSc consecutive patients, with no clinical evidence of cardiovascular involvement or pulmonary hypertension (PH), and 30 healthy controls (HC) in a retrospective, single-centre study. All SSc patients underwent recurrent clinical and echocardiographic assessments and NT-proANP plasma level was assessed at baseline. SSc-related cardiovascular events and deaths were extracted during a 6-year follow-up. The complete work-up for the diagnosis, treatment and management of PH performed along the 6 years of follow-up referred to the 2015 European Society of Cardiology guidelines. Results: Systemic sclerosis patients showed lower TAPSE/sPAP ratio at baseline compared to HC [SSc median value = 0.71 mm/mmHg, (IQR 0.62-0.88) vs. HC median value = 1.00 mm/mmHg, (IQR 0.96-1.05); p < 0.001]. Multivariable Cox analysis revealed TAPSE/sPAP ratio as an independent predictor for SSc-related cardiovascular events [HR = 3.436 (95% CI 1.577-7.448); p = 0.002] and mortality [HR = 3.653 (95% CI 1.712-8.892); p = 0.014]. The value of TAPSE/sPAP ratio < 0.7 mm/mmHg was identified as an optimal cut-off for predicting adverse outcomes (p < 0.001) by receiver operating characteristic (ROC) analyses. NT-proANP level significantly related to TAPSE/sPAP ratio (r = 0.52, p < 0.001). TAPSE/sPAP ratio combined with NT-proANP showed an overall significant prognostic role in this SSc population, confirmed by Kaplan-Meier analysis (Log rank p < 0.001). Conclusion: The TAPSE/sPAP ratio, as an index of RV/PA coupling, is an affordable predictor of cardiovascular events and mortality in SSc and, combined with NT-proANP level, may improve the clinical phenotyping and prognostic stratification of SSc patients.

Relevance of stromal interaction molecule 1 (STIM1) in experimental and human stroke.

Stroke represents a main cause of death and permanent disability worldwide. In the attempt to develop targeted preventive and therapeutic strategies, several efforts were performed over the last decades to identify the specific molecular abnormalities preceding cerebral ischemia and neuronal death. In this regard, mitochondrial dysfunction, autophagy, and intracellular calcium homeostasis appear important contributors to stroke development, as underscored by recent pre-clinical evidence. Intracellular calcium (Ca2+) homeostasis is regulated, among other mechanisms, by the calcium sensor stromal interaction molecule 1 (STIM1) and calcium release-activated calcium modulator (ORAI) members, which mediate the store-operated Ca2+ entry (SOCE). The activity of SOCE is deregulated in animal models of ischemic stroke, leading to ischemic injury exacerbation. We found a different pattern of expression of few SOCE components, dependent from a STIM1 mutation, in cerebral endothelial cells isolated from the stroke-prone spontaneously hypertensive rat (SHRSP), compared to the stroke-resistant (SHRSR) strain, suggesting a potential involvement of this mechanism into the stroke predisposition of SHRSP. In this article, we discuss the relevant role of STIM1 in experimental stroke, as highlighted by the current literature and by our recent experimental findings, and the available evidence in the human disease. We also provide a glance on future perspectives and clinical implications of STIM1.

Uncoupling Protein 2 as a Pathogenic Determinant and Therapeutic Target in Cardiovascular and Metabolic Diseases.

Uncoupling protein 2 (UCP2) is a mitochondrial protein that acts as an anion carrier. It is involved in the regulation of several processes, including mitochondrial membrane potential, generation of reactive oxygen species within the inner mitochondrial membrane and calcium homeostasis. UCP2 expression can be regulated at different levels: genetic (gene variants), transcriptional [by peroxisome proliferator-activated receptors (PPARs) and microRNAs], and post-translational. Experimental evidence indicates that activation of UCP2 expression through the AMPK/PPAR-α axis exerts a protective effect toward renal damage and stroke occurrence in an animal model of ischemic stroke (IS) associated with hypertension. UCP2 plays a key role in heart diseases (myocardial infarction and cardiac hypertrophy) and metabolic disorders (obesity and diabetes). In humans, UCP2 genetic variants (-866G/A and Ala55Val) associate with an increased risk of type 2 diabetes mellitus and IS development. Over the last few years, many agents that modulate UCP2 expression have been identified. Some of them are natural compounds of plant origin, such as Brassica oleracea, curcumin, berberine and resveratrol. Other molecules, currently used in clinical practice, include anti-diabetic (gliptin) and chemotherapeutic (doxorubicin and taxol) drugs. This evidence highlights the relevant role of UCP2 for the treatment of a wide range of diseases, which affect the national health systems of Western countries. We will review current knowledge on the physiological and pathological implications of UCP2 with particular regard to cardiovascular and metabolic disorders and will focus on the available therapeutic approaches affecting UCP2 level for the treatment of human diseases.

Role of DAMPs and of Leukocytes Infiltration in Ischemic Stroke: Insights from Animal Models and Translation to the Human Disease.

Stroke is a leading cause of death and disability worldwide. Several mechanisms are involved in the pathogenesis of ischemic stroke (IS). The contributory role of the inflammatory and immunity processes was demonstrated both in vitro and in animal models, and was confirmed in humans. IS evokes an immediate inflammatory response that involves complex cellular and molecular mechanisms. All components of the innate and adaptive immunity systems are involved in several steps of the ischemic cascade. In the early phase, inflammatory and immune mechanisms contribute to the brain tissue damage, whereas, in the late phase, they participate to the tissue repair processes. In particular, damage-associated molecular patterns (DAMPs) appear critical for the promotion of altered blood brain barrier permeability, leukocytes infiltration, tissue edema and brain injury. Conversely, the activation of regulatory T lymphocytes (Tregs) plays protective effects. The identification of specific cellular/molecular elements belonging to the inflammatory and immune responses, contributing to the brain ischemic injury and tissue remodeling, offers the advantage to design adequate therapeutic strategies. In this article, we will present an overview of the knowledge on inflammatory and immunity processes in IS, with a particular focus on the role of DAMPs and leukocytes infiltration. We will discuss evidence obtained in preclinical models of IS and in humans. The main molecular mechanisms useful for the development of novel therapeutic approaches will be highlighted. The translation of experimental findings to the human disease is still a difficult step to pursue. Further investigations are required to fill up the existing gaps.

An interplay between UCP2 and ROS protects cells from high-salt-induced injury through autophagy stimulation.

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.

Trehalose, a natural disaccharide, reduces stroke occurrence in the stroke-prone spontaneously hypertensive rat.

Cerebrovascular disease, a frequent complication of hypertension, is a major public health issue for which novel therapeutic and preventive approaches are needed. Autophagy activation is emerging as a potential therapeutic and preventive strategy toward stroke. Among usual activators of autophagy, the natural disaccharide trehalose (TRE) has been reported to be beneficial in preclinical models of neurodegenerative diseases, atherosclerosis and myocardial infarction. In this study, we tested for the first time the effects of TRE in the stroke-prone spontaneously hypertensive rat (SHRSP) fed with a high-salt stroke permissive diet (JD). We found that TRE reduced stroke occurrence and renal damage in high salt-fed SHRSP. TRE was also able to decrease systolic blood pressure. Through ex-vivo studies, we assessed the beneficial effect of TRE on the vascular function of high salt-fed SHRSP. At the molecular level, TRE restored brain autophagy and reduced mitochondrial mass, along with the improvement of mitochondrial function. The beneficial effects of TRE were associated with increased nuclear translocation of TFEB, a transcriptional activator of autophagy. Our results suggest that TRE may be considered as a natural compound efficacious for the prevention of hypertension-related target organ damage, with particular regard to stroke and renal damage.

Differential Expression of Sphingolipid Metabolizing Enzymes in Spontaneously Hypertensive Rats: A Possible Substrate for Susceptibility to Brain and Kidney Damage.

Alterations in the metabolism of sphingolipids, a class of biologically active molecules in cell membranes with direct effect on vascular homeostasis, are increasingly recognized as important determinant in different vascular disorders. However, it is not clear whether sphingolipids are implicated in the pathogenesis of hypertension-related cerebrovascular and renal damage. In this study, we evaluated the existence of possible abnormalities related to the sphingolipid metabolism in the brain and kidneys of two well validated spontaneously hypertensive rat strains, the stroke-prone (SHRSP) and the stroke-resistant (SHRSR) models, as compared to the normotensive Wistar Kyoto (WKY) rat strain. Our results showed a global alteration in the metabolism of sphingolipids in both cerebral and renal tissues of both hypertensive strains as compared to the normotensive rat. However, few defects, such as reduced expression of enzymes involved in the metabolism/catabolism of sphingosine-1-phosphate and in the de novo biosynthetic pathways, were exclusively detected in the SHRSP. Although further studies are necessary to fully understand the significance of these findings, they suggest that defects in specific lipid molecules and/or their related metabolic pathways may likely contribute to the pathogenesis of hypertensive target organ damage and may eventually serve as future therapeutic targets to reduce the vascular consequences of hypertension.

Beneficial effects of a combination of natural product activators of autophagy on endothelial cells and platelets.

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, H2 O2 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.

T2238C atrial natriuretic peptide gene variant and cardiovascular events in patients with atrial fibrillation: A substudy from the ATHERO-AF cohort.

BACKGROUND: The T2238C variant of the atrial natriuretic peptide (ANP) gene has emerged as a novel risk factor for the incidence of cardiovascular events. However, the impact of this variant on cardiovascular outcome in patients with atrial fibrillation (AF) is unknown. METHODS: We included 557 anticoagulated patients with non-valvular AF randomly selected from the prospective ATHERO-AF cohort. Patients underwent genetic analysis for the T2238C/ANP variant and were grouped as wild type or heterozygous or homozygous for C2238 variant allele. Primary endpoint was a composite of cardiovascular events (CVEs) including cardiovascular death, fatal/non-fatal ischemic stroke and myocardial infarction. Overall, 429 patients carried the TT wild type genotype, 110 patients (19.7%) were heterozygous (T/C) and 18 patients (3.2%) were homozygous (CC). RESULTS: Incidence of CVEs was higher in homozygous patients for C2238 allele at unadjusted analysis (log-rank test, p = 0.042 for additive model, p = 0.043 for recessive model). The multivariable Cox proportional hazards regression analysis confirmed that C2238 ANP allele was associated with CVEs in the additive (p = 0.008) and recessive models (p = 0.005). CONCLUSIONS: Carrier status for the C2238/ANP variant allele is associated with an increased risk of CVEs in anticoagulated AF patients.

Real-life appraisal on blood pressure targets achievement in adult outpatients at high cardiovascular risk.

BACKGROUND AND AIM: Although hypertension guidelines highlight the benefits of achieving the recommended blood pressure (BP) targets, hypertension control rate is still insufficient, mostly in high or very high cardiovascular (CV) risk patients. Thus, we aimed to estimate BP control in a cohort of patients at high CV risk in both primary and secondary prevention. METHODS AND RESULTS: A single-center, cross-sectional study was conducted by extracting data from a medical database of adult outpatients aged 40-75 years, who were referred to our Hypertension Unit, Rome (IT), for hypertension assessment. Office BP treatment targets were defined according to 2018 ESC/ESH guidelines as: a)<130/80 mmHg in individuals aged 40-65 years; b)<140/80 mmHg in subjects aged >65 years. Primary prevention patients with SCORE <5% were considered to be at low-intermediate risk, whilst individuals with SCORE ≥5% or patients with comorbidities were defined to be at very high risk. Among 6354 patients (47.2% female, age 58.4 ± 9.6 years), 4164 (65.5%) were in primary prevention with low-intermediate CV risk, 1831 (28.8%) in primary prevention with high-very high CV risk and 359 (5.6%) in secondary prevention. In treated hypertensive outpatients, uncontrolled hypertension rate was significantly higher in high risk primary prevention than in low risk primary prevention and secondary prevention patients (18.4% vs 24.4% vs. 12.5%, respectively; P < 0.001). In high risk primary prevention diabetic patients only 10% achieved the recommended BP targets. CONCLUSIONS: Our data confirmed unsatisfactory BP control among high-risk patients, both in primary and secondary prevention, and suggest the need for a more stringent BP control policies in these patients.