Molecular Implications of natriuretic peptides in the protection from hypertension and target organ damage development

The pathogenesis of hypertension, as a multifactorial trait, is complex. High blood pressure levels, in turn, concur with the development of cardiovascular damage. Abnormalities of several neurohormonal mechanisms controlling blood pressure homeostasis and cardiovascular remodeling can contribute to these pathological conditions. The natriuretic peptide (NP) family (including ANP (atrial natriuretic peptide), BNP (brain natriuretic peptide), and CNP (C-type natriuretic peptide)), the NP receptors (NPRA, NPRB, and NPRC), and the related protease convertases (furin, corin, and PCSK6) constitute the NP system and represent relevant protective mechanisms toward the development of hypertension and associated conditions, such as atherosclerosis, stroke, myocardial infarction, heart failure, and renal injury. Initially, several experimental studies performed in different animal models demonstrated a key role of the NP system in the development of hypertension. Importantly, these studies provided relevant insights for a better comprehension of the pathogenesis of hypertension and related cardiovascular phenotypes in humans. Thus, investigation of the role of NPs in hypertension offers an excellent example in translational medicine. In this review article, we will summarize the most compelling evidence regarding the molecular mechanisms underlying the physiological and pathological impact of NPs on blood pressure regulation and on hypertension development. We will also discuss the protective effect of NPs toward the increased susceptibility to hypertensive target organ damage

The T2238C human atrial natriuretic peptide molecular variant and the risk of cardiovascular diseases

Atrial natriuretic peptide (ANP) is a cardiac hormone which plays important functions to maintain cardio-renal homeostasis. The peptide structure is highly conserved among species. However, a few gene variants are known to fall within the human ANP gene. The variant rs5065 (T2238C) exerts the most substantial effects. The T to C transition at the 2238 position of the gene (13-23% allele frequency in the general population) leads to the production of a 30-, instead of 28-, amino-acid-long α-carboxy-terminal peptide. In vitro, CC2238/αANP increases the levels of reactive oxygen species and causes endothelial damage, vascular smooth muscle cells contraction, and increased platelet aggregation. These effects are achieved through the deregulated activation of type C natriuretic peptide receptor, the consequent inhibition of adenylate cyclase activity, and the activation of Giα proteins. In vivo, endothelial dysfunction and increased platelet aggregation are present in human subjects carrying the C2238/αANP allele variant. Several studies documented an increased risk of stroke and of myocardial infarction in C2238/αANP carriers. Recently, an incomplete response to antiplatelet therapy in ischemic heart disease patients carrying the C2238/αANP variant and undergoing percutaneous coronary revascularization has been reported. In summary, the overall evidence supports the concept that T2238C/ANP is a cardiovascular genetic risk factor that needs to be taken into account in daily clinical practice

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

Reduced brain UCP2 expression mediated by microRNA-503 contributes to increased stroke susceptibility in the high-salt fed stroke-prone spontaneously hypertensive rat

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

Functional Role of Natriuretic Peptides in Risk Assessment and Prognosis of Patients with Mitral Regurgitation

The management of mitral valve regurgitation (MR), a common valve disease, represents a challenge in clinical practice, since the indication for either surgical or percutaneous valve replacement or repair are guided by symptoms and by echocardiographic parameters which are not always feasible. In this complex scenario, the use of natriuretic peptide (NP) levels would serve as an additive diagnostic and prognostic tool. These biomarkers contribute to monitoring the progression of the valve disease, even before the development of hemodynamic consequences in a preclinical stage of myocardial damage. They may contribute to more accurate risk stratification by identifying patients who are more likely to experience death from cardiovascular causes, heart failure, and cardiac hospitalizations, thus requiring surgical management rather than a conservative approach. This article provides a comprehensive overview of the available evidence on the role of NPs in the management, risk evaluation, and prognostic assessment of patients with MR both before and after surgical or percutaneous valve repair. Despite largely positive evidence, a series of controversial findings exist on this relevant topic. Recent clinical trials failed to assess the role of NPs following the interventional procedure. Future larger studies are required to enable the introduction of NP levels into the guidelines for the management of MR

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

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

Dickkopf-3 upregulates VEGF in cultured human endothelial cells by activating activin receptor-like kinase 1 (ALK1) pathway

Dkk-3 is a member of the dickkopf protein family of secreted inhibitors of the Wnt pathway, which has been shown to enhance angiogenesis. The mechanism underlying this effect is currently unknown. Here, we used cultured HUVECs to study the involvement of the TGF-β and VEGF on the angiogenic effect of Dkk-3. Addition of hrDkk-3 peptide (1 or 10 ng/ml) to HUVECs for 6 or 12 h enhanced the intracellular and extracellular VEGF protein levels, as assessed by RTPCR, immunoblotting, immunocytochemistry and ELISA. The increase in the extracellular VEGF levels was associated to the VEGFR2 activation. Pharmacological blockade of VEGFR2 abrogated Dkk-3-induced endothelial cell tubes formation, indicating that VEGF is a molecular player of the angiogenic effects of Dkk-3. Moreover, Dkk-3 enhanced Smad1/5/8 phosphorylation and recruited Smad4 to the VEGF gene promoter, suggesting that Dkk-3 activated ALK1 receptor leading to a transcriptional activation of VEGF. This mechanism was instrumental to the increased VEGF expression and endothelial cell tubes formation mediated by Dkk-3, because both effects were abolished by siRNA-mediated ALK1 knockdown. In summary, we have found that Dkk-3 activates ALK1 to stimulate VEGF production and induce angiogenesis in HUVECs

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

A Decrease of Brain MicroRNA-122 Level Is an Early Marker of Cerebrovascular Disease in the Stroke-Prone Spontaneously Hypertensive Rat

Based on preliminary evidence that highlights microRNA-122 as a contributing factor to stroke pathogenesis, we aimed at assessing its expression level, along with the presence of early signs of cerebrovascular disease, in the brain of stroke-prone spontaneously hypertensive rat (SHRSP), a suitable model of human disease that accelerates stroke occurrence under a high sodium/low potassium (Japanese-style) diet (JD). After one month of JD, before stroke occurrence, brain microRNA-122 level was significantly decreased in SHRSP as compared to the stroke-resistant SHR (SHRSR). At this time, levels of markers of oxidative stress and inflammation, as well as of endothelial integrity and function, apoptosis and necrosis were differently modulated in the brains of JD-fed SHRSP as compared to SHRSR, pointing to a significant activation of all deleterious mechanisms underlying subsequent stroke development in SHRSP. We also showed that miR-122 improved survival of rat endothelial cerebral cells upon stress stimuli (excess NaCl, hydrogen peroxide). Our data suggest that a decrease of brain microRNA-122 level is deleterious and can be considered as an early marker of stroke in the SHRSP. Understanding the mechanisms by which microRNA-122 protects vascular cells from stress stimuli may provide a useful approach to improve preventive and treatment strategies against stroke