Maria Cotugno

Differential Modulation of Uncoupling Protein 2 in Kidneys of Stroke-Prone Spontaneously Hypertensive Rats Under High-Salt/Low-Potassium Diet

The stroke-prone spontaneously hypertensive rat (SHRsp) represents an animal model of increased susceptibility to high-salt diet–induced cerebral and renal vascular injuries. High blood pressure and genetic factors are viewed as major contributing factors. In high-salt–loaded SHRsp and stroke-resistant SHR animals, we determined blood pressure levels, degree of kidney lesions, renal uncoupling protein 2 (UCP2) gene and protein expression levels along with rattus norvegicus (rno)-microRNA (miR) 24 and 34a gene expression, nuclear factor-&kgr;B protein levels, and oxidative stress. In vitro, UCP2 gene silencing was performed in renal mesangial cells. We found more severe degree of renal damage in SHRsp at the end of 4-week high-salt dietary treatment as compared with stroke-resistant SHR, despite comparable blood pressure levels, along with increased rate of inflammation and oxidative stress. Kidney UCP2 gene and protein expression levels were significantly downregulated under high-salt diet in SHRsp, but not in stroke-resistant SHR. Differential UCP2 regulation was paralleled by differential expression of kidney rno-miR 24 and 34a, known to target UCP2 gene, in the 2 strains. UCP2 gene silencing in renal mesangial cells led to increased rate of reactive oxygen species generation, increased inflammation and apoptosis, reduced cell vitality, and increased necrosis. In conclusion, high-salt diet downregulates the antioxidant UCP2-dependent mechanism in kidneys of SHRsp, but not of stroke-resistant SHR. A parallel differential kidney miR regulation under high-salt diet in the 2 strains may contribute to the differential UCP2 modulation. UCP2 is a critical protein to prevent oxidative stress damage in renal mesangial cells in vitro.

Influence of rs5065 atrial natriuretic peptide gene variant on coronary artery disease

OBJECTIVES 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. BACKGROUND Either modified ANP plasma levels or peptide structural alterations have been involved in development of cardiovascular events. METHODS 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. RESULTS 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. CONCLUSIONS The MA of rs5065 ANP gene variant associates with increased susceptibility to ACS and has unfavorable prognostic value in CAD.

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-&agr;ANP. Methods and Results: In vitro, human umbilical vein endothelial cell were exposed to either wild-type (T2238)- or mutant (C2238)-&agr;ANP. Cell survival and apoptosis were tested by Trypan blue, annexin V, and cleaved caspase-3 assays. C2238-&agr;ANP significantly reduced human umbilical vein endothelial cell survival and increased apoptosis. In addition, C2238-&agr;ANP reduced endothelial tube formation, as assessed by matrigel. C2238-&agr;ANP did not differentially modulate natriuretic peptide receptor (NPR)-A/B activity with respect to T2238-&agr;ANP, as evaluated by intracellular cGMP levels. In contrast, C2238-&agr;ANP, but not T2238-&agr;ANP, markedly reduced intracellular cAMP levels in an NPR-C–dependent manner. Accordingly, C2238-&agr;ANP showed higher affinity binding to NPR-C, than T2238-&agr;ANP. 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-&agr;ANP. Similar data were obtained in human aortic endothelial cell with NPR-C knockdown. NPR-C activation by C2238-&agr;ANP inhibited the protein kinase A/Akt1 pathway and increased reactive oxygen species. Adenovirus-mediated Akt1 reactivation rescued the detrimental effects of C2238-&agr;ANP. Overall, these data indicate that C2238-&agr;ANP 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-&agr;ANP 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-&agr;ANP 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.

Ndufc2 Gene Inhibition Is Associated With Mitochondrial Dysfunction and Increased Stroke Susceptibility in an Animal Model of Complex Human Disease

Background 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. Methods and Results 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). Conclusions 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.

Association of a single nucleotide polymorphism of the NPR3 gene promoter with early onset ischemic stroke in an Italian cohort.

BACKGROUND NPR3, located on human chromosome 5 (5p14-p13), encodes the natriuretic peptide receptor type C (NPR-C) that is mainly known as the natriuretic peptide clearance receptor. Involvement of NPR3 in susceptibility to cardiovascular diseases, i.e. hypertension, has been previously shown. With regard to stroke predisposition, evidence for a potential role of genetic variation within or nearby NPR3 has been suggested by a previous genome wide association study. METHODS We investigated the contribution to early-onset ischemic stroke susceptibility of the NPR3 -55 C>A transition by genotyping this variant in an Italian cohort of 368 cases and 335 controls. RESULTS In a multivariable logistic regression analysis adjusting for age, gender, hypertension, hypercholesterolemia, smoking habit and diabetes, a significant association of the -55 AA genotype with stroke was observed (OR=3.2, 95% CI 1.2-8.3, p=0.016). Remarkably, the polymorphism remained associated with stroke after adjusting for hypertensive status. CONCLUSION Our observation obtained in an Italian cohort of early onset ischemic strokes suggests that a NPR3 promoter gene variant could have a role on cerebrovascular disease susceptibility.

Protective effects of Brassica oleracea sprouts extract toward renal damage in high-salt-fed SHRSP: role of AMPK/PPARa/UCP2 axis

Objectives: Renal damage precedes occurrence of stroke in high-sodium/low-potassium-fed stroke-prone spontaneously hypertensive rat (SHRSP). We previously reported a marked suppression of uncoupling protein-2 (UCP2) upon high-salt Japanese-style diet in SHRSP kidneys. Vegetable compounds are known to exert protective effects in cardiovascular diseases. We aimed at evaluating the impact of Brassica oleracea sprouts juice toward renal damage in Japanese diet-fed SHRSP and exploring the role of 5′-adenosine monophosphate-activated protein kinase (AMPK)/NAD-dependent deacetylase sirtuin-1 (SIRT1)/peroxisome proliferator-activated receptor-&ggr; coactivator-1&agr; (PGC1&agr;)/peroxisome proliferator-activated receptor-&agr; (PPAR&agr;)/UCP2 axis. Methods: SHRSP received Japanese diet for 4 weeks. A group of SHRSP received Japanese diet and B. oleracea. A third group received Japanese diet, B. oleracea, and PPAR&agr; inhibitor (GW6471). A group of SHRSP fed with regular diet served as control. Results: Japanese diet induced marked increases of oxidative stress, inflammation, and proteinuria, along with glomerular and tubular damage, as compared with regular diet. A significant suppression of AMPK/UCP2 pathway was observed. Despite Japanese diet feeding, concomitant administration of B. oleracea prevented oxidative stress accumulation, inflammation, renal damage, and proteinuria. All components of the UCP2 regulatory pathway were significantly increased by B. oleracea. Superoxide dismutase 2 and phosphoendothelial nitric oxide synthase were also stimulated. Addition of PPAR&agr; inhibitor to B. oleracea and Japanese diet significantly reduced the B. oleracea beneficial effects. SBP levels were comparable among the different groups of rats. In vitro, UCP2 inhibition by genipin offset the antioxidant effect of B. oleracea in renal mesangial and proximal tubular cells. Conclusion: B. oleracea administration prevented renal damage in salt-loaded SHRSP, independently from SBP, with parallel stimulation of AMPK/SIRT1/PGC1&agr;/PPAR&agr;/UCP2 axis. Stimulation of the latter mechanism may provide relevant renal protective effect and play a therapeutic role in target organ damage progression in hypertension.

Aminoterminal natriuretic peptides and cardiovascular risk in an Italian male adult cohort

adult cohort☆,☆☆ Antonio Barbato , Sebastiano Sciarretta , Simona Marchitti , Roberto Iacone , Sara Di Castro , Rosita Stanzione , Maria Cotugno , Renato Ippolito , Luigi Palmieri , Camilla Calvieri , Allegra Battistoni , Massimo Volpe , Pasquale Strazzullo , Speranza Rubattu b,c,⁎ and On behalf of the Olivetti Heart Study Research Group a Department of Clinical and Experimental Medicine, Federico II University of Naples Medical School, Naples, Italy b IRCCS Neuromed, Pozzilli (Is) Italy c Department of Cardiology, School of Medicine and Psychology, University Sapienza of Rome, Ospedale S. Andrea, Rome, Italy d Department of Cerebro and Cardiovascular Diseases Epidemiology, Istituto Superiore di Sanita, Rome, Italy

NT-proANP circulating level is a prognostic marker in stable ischemic heart disease

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C2238/αANP modulates apolipoprotein E through Egr-1/miR199a in vascular smooth muscle cells in vitro

Subjects carrying the T2238C ANP gene variant have a higher risk to suffer a stroke or myocardial infarction. The mechanisms through which T2238C/αANP exerts detrimental vascular effects need to be fully clarified. In the present work we aimed at exploring the impact of C2238/αANP (mutant form) on atherosclerosis-related pathways. As a first step, an atherosclerosis gene expression macroarray analysis was performed in vascular smooth muscle cells (VSMCs) exposed to either T2238/αANP (wild type) or C2238/αANP. The major finding was that apolipoprotein E (ApoE) gene expression was significantly downregulated by C2238/αANP and it was upregulated by T2238/αANP. We subsequently found that C2238/αANP induces ApoE downregulation through type C natriuretic peptide receptor (NPR-C)-dependent mechanisms involving the upregulation of miR199a-3p and miR199a-5p and the downregulation of DNAJA4. In fact, NPR-C knockdown rescued ApoE level. Upregulation of miR199a by NPR-C was mediated by a reactive oxygen species-dependent increase of the early growth response protein-1 (Egr-1) transcription factor. In fact, Egr-1 knockdown abolished the impact of C2238/αANP on ApoE and miR199a. Of note, downregulation of ApoE by C2238/αANP was associated with a significant increase in inflammation, apoptosis and necrosis that was completely rescued by the exogenous administration of recombinant ApoE. In conclusion, our study dissected a novel mechanism of vascular damage exerted by C2238/αANP that is mediated by ApoE downregulation. We provide the first demonstration that C2238/αANP downregulates ApoE in VSMCs through NPR-C-dependent activation of Egr-1 and the consequent upregulation of miR199a. Restoring ApoE levels could represent a potential therapeutic strategy to counteract the harmful effects of C2238/αANP.

The C2238/αANP variant is a negative modulator of both viability and function of coronary artery smooth muscle cells.

Background Abnormalities of vascular smooth muscle cells (VSMCs) contribute to development of vascular disease. Atrial natriuretic peptide (ANP) exerts important effects on VSMCs. A common ANP molecular variant (T2238C/αANP) has recently emerged as a novel vascular risk factor. Objectives We aimed at identifying effects of CC2238/αANP on viability, migration and motility in coronary artery SMCs, and the underlying signaling pathways. Methods and Results Cells were exposed to either TT2238/αANP or CC2238/αANP. At the end of treatment, cell viability, migration and motility were evaluated, along with changes in oxidative stress pathway (ROS levels, NADPH and eNOS expression), on Akt phosphorylation and miR21 expression levels. CC2238/αANP reduced cell vitality, increased apoptosis and necrosis, increased oxidative stress levels, suppressed miR21 expression along with consistent changes of its molecular targets (PDCD4, PTEN, Bcl2) and of phosphorylated Akt levels. As a result of increased oxidative stress, CC2238/αANP markedly stimulated cell migration and increased cell contraction. NPR-C gene silencing with specific siRNAs restored cell viability, miR21 expression, and reduced oxidative stress induced by CC2238/αANP. The cAMP/PKA/CREB pathway, driven by NPR-C activation, significantly contributed to both miR21 and phosphoAkt reduction upon CC2238/αANP. miR21 overexpression by mimic-hsa-miR21 rescued the cellular damage dependent on CC2238/αANP. Conclusions CC2238/αANP negatively modulates viability through NPR-C/cAMP/PKA/CREB/miR21 signaling pathway, and it augments oxidative stress leading to increased migratory and vasoconstrictor effects in coronary artery SMCs. These novel findings further support a damaging role of this common αANP variant on vessel wall and its potential contribution to acute coronary events.