Rosita Stanzione

Natriuretic peptides: An update on bioactivity, potential therapeutic use, and implication in cardiovascular diseases

The natriuretic peptide system includes three known peptides: atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and C-type natriuretic peptide (CNP). They contribute to the regulation of cardiovascular homeostasis through diuretic, natriuretic, and vasodilatory properties. Among them, ANP has received particular attention because of its effects on blood pressure regulation and cardiac function. Although the potential for its therapeutic application in the treatment of hypertension and heart failure has been evaluated in several experimental and clinical investigations, no pharmacological approach directly targeted at modulation of ANP levels has ever reached the stage of being incorporated into clinical practice. Recently, ANP has also received attention as being a possible cardiovascular risk factor, particularly in the context of hypertension, stroke, obesity, and metabolic syndrome. Abnormalities in either peptide levels or peptide structure are thought to underlie its implied role in mediating cardiovascular diseases. Meanwhile, BNP has emerged as a relevant marker of left ventricular (LV) dysfunction and as a useful predictor of future outcome in patients with heart failure. This review deals with the major relevant findings related to the cardiovascular and metabolic effects of natriuretic peptides, to their potential therapeutic use, and to their role in mediating cardiovascular diseases.

Atrial Natriuretic Peptide Gene Polymorphisms and Risk of Ischemic Stroke in Humans

Background and Purpose— A precise definition of genetic factors responsible for common forms of stroke is still lacking. The purpose of the present study was to investigate the contributory role of the genes encoding atrial natriuretic peptide (ANP) and type A natriuretic peptide receptor (NPRA) in humans’ susceptibility to develop ischemic stroke. Methods— Allele and genotype frequencies of ANP and NPRA were characterized in an Italian case-control study with patients affected by vascular disease or risk factors. Subjects were recruited from the island of Sardinia (206 cases, 236 controls). Results— A significant association between the ANP/TC2238 polymorphic site and stroke occurrence was found when a recessive model of inheritance was assumed. The risk conferred by this mutant genotype, when estimated by multivariate logistic regression analysis, was 3.8 (95% confidence interval, 1.4 to 10.9). A significantly increased risk of stroke recurrence was observed among cases carrying the ANP/CC2238 genotype compared with cases carrying the ANP/TT2238 genotype (P =0.04). No direct association of NPRA with stroke occurrence was detected. However, a significant epistatic interaction between the ANP/CC2238 genotype and an allelic variant of NPRA led to a 5.5-fold increased risk of stroke (95% confidence interval, 1.5 to 19.4). Conclusions— Our findings support a direct contributory role of ANP to stroke in humans. A significant interaction between ANP and NPRA on stroke occurrence was found.

Gene polymorphisms of the renin-angiotensin-aldosterone system and the risk of ischemic stroke: a role of the A1166C/AT1 gene variant.

Objective The role of the renin–angiotensin–aldosterone system (RAAS) genes on predisposition to develop stroke, a multifactorial and polygenic cardiovascular trait, is still under investigation. In the present study we characterized the contributory role of RAAS genes in the susceptibility to develop ischemic stroke in humans. Methods Allele and genotype frequencies of RAAS genes were characterized in a population of 215 cases (including only atherothrombotic and lacunar forms) and 236 controls selected in Sardinia, a large Mediterranean island with a well-known segregated population. Statistical analysis was performed in the whole population and, based on a significant interaction between angiotensin II receptor (AT1) genotype and hypertension, was also repeated in the hypertensive subgroup. Results A significant association of the C1166/AT1 gene allelic variant with stroke was found when assuming a dominant model of transmission [unadjusted odds ratio (OR) = 1.5, 95% confidence interval (CI) 1.1–2.2, P = 0.024]. The strength of the association became more evident in the subgroup of hypertensive individuals (135 cases and 110 controls). In fact, in this cohort the independent OR for the AT1 gene was 2.1, 95% CI 1.2–3.7, P = 0.006 in the dominant model and 2.0, 95% CI 1.3–3.2, P = 0.002 in the additive model. No other RAAS gene was identified as a contributor to stroke. Conclusions Our findings support a predisposing role of an AT1 gene variant in the development of ischemic stroke. In particular, the AT1 gene variant exerted a major impact on ischemic stroke occurrence in the presence of hypertension.

Contribution of Genetic Factors to Renal Lesions in the Stroke-Prone Spontaneously Hypertensive Rat

Abstract—Stroke-prone spontaneously hypertensive rats (SHRSP) develop renal lesions more frequently than the closely related control strain, the stroke-resistant SHR. The aim of this study was to investigate the contribution of genetic factors to the enhanced susceptibility to renal damage of SHRSP in an SHRSP/SHR F2 intercross by means of a genotype/phenotype cosegregation analysis. For this purpose, 154 6-week-old F2-SHRSP/SHR rats (79 male, 75 female) were fed a stroke-permissive Japanese diet for 4 weeks. Systolic blood pressure (SBP) was recorded at the end of the dietary period. Renal damage was scored from 0 to 3, and 274 genetic markers polymorphic between SHR and SHRSP were genotyped. Linkage of genotype markers to the degree of renal disease was determined by &khgr;2 test. Experimental threshold level to declare linkage was calculated by QTL cartographer. SBP was not correlated to renal damage (&rgr; coefficient, 0.201; P =NS). Grade 2 and grade 3 lesions were more frequent in male than in female rats (P =0.01). Two loci, D1Rat238, on chromosome 1 and the IGF receptor-binding protein 4 (Rbp4g) on chromosome 10, were significantly linked to the degree of renal damage, with SHRSP allele being aggressive at D1Rat238 locus and protective at Rbp4g locus. In male rats only, the SHRSP allele at one locus on chromosome 16, D16Mit2, was associated with a more severe degree of renal disease. Our results demonstrate that in this intercross, susceptibility to renal damage is influenced by several genetic loci acting independently from high blood pressure levels and also shows a sexual dimorphism.

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.

Polymorphisms in prothrombotic genes and their impact on ischemic stroke in a Sardinian population

Genetic factors are involved in the individual predisposition to develop ischemic stroke (IS). In the present study we tested the role of the Factor VII G10976A and -C122T polymorphisms on the susceptibility to develop IS in a genetically homogenous and clinically well ascertained case-control study including 294 cases (median age 75 years; 176 males/118 females) and 286 controls (median age 73 years; 163 males/123 females) in Sardinia, Italy. In addition, we carried out an exploratory analysis with respect to other frequently studied polymorphisms of haemostatic factor genes:Factor II G20210A, Factor V G1691A,,Fibrinogen alpha-chain Thr312Ala, Fibrinogen beta-chain -C148T, Factor XIII G185T, GPIIb/IIIa T1565C. Among all the genes tested, FVII -C122T showed a significant, independent contribution to IS predisposition both in crude and adjusted analyses (crude OR 1.52, 95% CI 1.09-2.10, P=0.013; adjusted OR 1.48, 95% CI 1.04-2.09, P=0.028, respectively). Haplotype analyses revealed a conserved population structure with high linkage disequilibrium between both FVII mutations tested. Blood levels of FVII had an inverse relationship with the polymorphism involved. Apart from genetic influence, there was a significant role for hypertension (OR=1.7, 95% CI 1.19-2.43, P=0.003), hypercholesterolemia (OR=2.21, 95% CI 1.38-3.54, P=0.001) and atrial fibrillation (OR=1.66, 95% CI 1.06-2.58, P=0.026) on IS occurrence. In summary, we describe evidence for a possible direct association of FVII gene molecular variants with the occurrence of IS in a genetically homogenous human sample.

Phosphodiesterase 4D and 5-lipoxygenase activating protein genes and risk of ischemic stroke in Sardinians

Genetic factors contribute to the risk of ischemic stroke (IS). The phosphodiesterase-4D (PDE4D) and the 5-lipoxygenase activating protein (ALOX5AP) genes were identified as contributors to stroke in an Icelandic population. In an attempt to better define the contributory role of PDE4D and ALOX5AP genes to the risk of IS in humans, we carried out the present association study in a well-characterized, earlier published, genetically homogenous population from the island of Sardinia, Italy. In this cohort, including 294 cases and 235 controls, age, hypertension, hypercholesterolemia, and atrial fibrillation represent risk factors for IS. The PDE4D gene was evaluated by four single nucleotide polymorphisms (SNP32, SNP45, SNP83, SNP87) and by the microsatellite AC008818-1; the ALOX5AP gene was characterized by three SNPs (SG13S32, SG13S89, ALO2A). The results of our study provide no evidence of association between any single PDE4D and ALOX5AP gene variant with the risk of IS in the Sardinian cohort. Haplotype analysis, including that constructed with allele 0 of microsatellite AC008818-1 and SNP45 of the PDE4D gene, was also negative. In conclusion, we found no evidence of association between PDE4D and ALOX5AP genes and the risk of IS in a genetically homogenous population from Sardinia.

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.