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.

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.

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

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

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.

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.

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.

Epigenetic control of natriuretic peptides: implications for health and disease.

The natriuretic peptides (NPs) family, including a class of hormones and their receptors, is largely known for its beneficial effects within the cardiovascular system to preserve regular functions and health. The concentration level of each component of the family is of crucial importance to guarantee a proper control of both systemic and local cardiovascular functions. A fine equilibrium between gene expression, protein secretion and clearance is needed to achieve the final optimal level of NPs. To this aim, the regulation of gene expression and translation plays a key role. In this regard, we know the existence of fine regulatory mechanisms, the so-called epigenetic mechanisms, which target many genes at either the promoter or the 3'UTR region to inhibit or activate their expression. The gene encoding ANP (NPPA) is regulated by histone modifications, DNA methylation, distinct microRNAs and a natural antisense transcript (NPPA-AS1) with consequent implications for both health and disease conditions. Notably, ANP modulates microRNAs on its own. Histone modifications of BNP gene (NPPB) are associated with several cardiomyopathies. The proBNP processing is regulated by miR30-GALNT1/2 axis. Among other components of the NPs family, CORIN, NPRA, NPRC and NEP may undergo epigenetic regulation. A better understanding of the epigenetic control of the NPs family will allow to gain more insights on the pathological basis of common cardiovascular diseases and to identify novel therapeutic targets. The present review article aims to discuss the major achievements obtained so far with studies on the epigenetic modulation of the NPs family.

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.

Brain Overexpression of Uncoupling Protein-2 (UCP2) Delays Renal Damage and Stroke Occurrence in Stroke-Prone Spontaneously Hypertensive Rats.

The downregulation of uncoupling protein-2 (UCP2) is associated with increased brain and kidney injury in stroke-prone spontaneously hypertensive rats (SHRSP) fed with a Japanese style hypersodic diet (JD). Systemic overexpression of UCP2 reduces organ damage in JD-fed SHRSP. We examined the effect of brain-specific UCP2 overexpression on blood pressure (BP), stroke occurrence and kidney damage in JD-fed SHRSP. Rats received a single i.c.v. injection of a lentiviral vector encoding UCP2 (LV-UCP2), or an empty vector. The brain delivery of LV-UCP2 significantly delayed the occurrence of stroke and kidney damage. The large reduction of proteinuria observed after LV-UCP2 injection was unexpected, because BP levels were unchanged. At the time of stroke, rats treated with LV-UCP2 still showed a large UCP2 upregulation in the striatum, associated with increases in OPA1 and FIS1 protein levels, and reductions in PGC1-α, SOD2, TNFα mRNA levels and NRF2 protein levels. This suggested UCP2 overexpression enhanced mitochondrial fusion and fission and reduced oxidative damage and inflammation in the striatum of JD-fed SHRSP rats. Our data suggest the existence of central mechanisms that may protect against hypertension-induced organ damage independently of BP, and strengthen the suitability of strategies aimed at enhancing UCP2 expression for the treatment of hypertensive damage.

RyR2 Common Gene Variant G1886S and the Risk of Ventricular Arrhythmias in ICD Patients with Heart Failure.

BACKGROUND: Cardiac ryanodine receptor 2 (RyR2) is critical to the electrical homeostasis of cardiomyocytes. Its gene variant rs3766871 entails channel destabilization and enhanced intracellular Ca(2+) oscillation, thus promoting cardiac arrhythmias. We investigated whether the RyR2 rs3766871 variant is associated with aborted sudden cardiac death or ICD therapy for ventricular tachycardia (VT)/fibrillation (VF) in heart failure (HF) patients implanted with a cardioverter defibrillator (ICD). METHODS AND RESULTS: A total of 183 HF patients with primary or secondary prevention ICD were divided in 2 groups. A VT/VF group was composed of secondary prevention patients and primary prevention patients with appropriate ICD intervention for VT/VF. An ICD control group was composed of primary prevention patients free from any appropriate ICD intervention after 43 ± 25 months follow-up. Study subjects were genotyped with respect to the rs3766871 RyR2 gene variant. Hazard ratios (HRs) were derived from Cox proportional-hazards regression analysis. In all, 56 patients constituted the VT/VF group and 127 patients the ICD control group. Male sex (HR: 3.02; 95% CI: 0.99-9.18; P = 0.05), atrial fibrillation (AF; HR: 2.33; 95% CI: 0.89-6.10; P = 0.08), and underuse of ß-blockers (HR: 2.08; 95% CI: 0.84-5.15; P = 0.11) were associated with the VT/VF phenotype. Prevalence of the rs3766871 minor allele was 2.8% in ICD control patients and 8.0% in the VT/VF group (P = 0.02). After adjustment for age, sex, AF, and use of ß-blockers, the rs3766871 minor allele was associated with increased risk of VT/VF (HR: 3.49; 95% CI: 1.14-10.62; P = 0.02). CONCLUSIONS: Our study identifies a significant role of RyR2 rs3766871 minor allele for increased susceptibility to VT/VF in a population of ICD patients with HF.

C2238 atrial natriuretic peptide molecular variant is associated with endothelial damage and dysfunction through natriuretic peptide receptor C signaling.

RATIONALE: C2238 atrial natriuretic peptide (ANP) minor allele (substitution of thymidine with cytosine in position 2238) associates with increased risk of cardiovascular events. OBJECTIVE: We investigated the mechanisms underlying the vascular effects of C2238-αANP. METHODS AND RESULTS: 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. CONCLUSIONS: 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.

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

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

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

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

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

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

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.

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.

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.