Tommaso Prescimone

Expression of C-type natriuretic peptide and its receptor NPR-B in cardiomyocytes

C-type natriuretic peptide (CNP) was recently found in myocardium at the mRNA and protein levels, but it is not known whether cardiomyocytes are able to produce CNP. The aim of this study was to determine the expression of CNP and its specific receptor NPR-B in cardiac cells, both in vitro and ex vivo. CNP, brain natriuretic peptide (BNP) and natriuretic peptide receptor (NPR)-B mRNA expression were examined by RT-PCR in the H9c2 rat cardiac myoblast cell line, in neonatal rat primary cardiomyocytes and in human umbilical vein endothelial cells (HUVECs) as control. CNP protein expression was probed in cardiac tissue sections obtained from adult male minipigs by immunohistochemistry, and in H9c2 cells both by immunocytochemistry and by specific radioimmunoassay. The results showed that cardiac cells as well as endothelial cells were able to produce CNP. Unlike cardiomyocytes, as expected, in endothelial cells expression of BNP was not detected. NPR-B mRNA expression was found in both cell types. Production of CNP in the heart muscle cells at protein level was confirmed by radioimmunological determination (H9c2: CNP=0.86 ± 0.083 pg/mg) and by immunocytochemistry studies. By immunostaining of tissue sections, CNP was detected in both endothelium and cardiomyocytes. Expression of CNP in cardiac cells at gene and protein levels suggests that the heart is actively involved in the production of CNP.

Selection of reference genes for normalization of real-time PCR data in minipig heart failure model and evaluation of TNF-α mRNA expression

Real-time PCR is the benchmark method for measuring mRNA expression levels, but the accuracy and reproducibility of its data greatly depend on appropriate normalization strategies. Though the minipig model is largely used to study cardiovascular disease, no specific reference genes have been identified in porcine myocardium. The aim of the study was to identify and validate reference gene to be used in RT-PCR studies of failing (HF) and non-failing pig hearts. Eight candidate reference genes (GAPDH, ACTB, B2M, TBP, HPRT-1, PPIA, TOP2B, YWHAZ) were selected to compare cardiac tissue of normal (n=4) and HF (n=5) minipigs. The most stable genes resulted: HPRT-1, TBP, PPIA (right and left atrium); PPIA, GAPDH, ACTB (right ventricle); HPRT-1, TBP, GAPDH (left ventricle). The normalization strategy was tested analyzing mRNA expression of TNF-α, which is known to be up-regulated in HF and whose variations resulted more significant when normalized with the appropriately selected reference genes. The findings obtained in this study underline the importance to provide a set of reference genes to normalize mRNA expression in HF and control minipigs. The use of unvalidated reference genes can generate biased results because also their expression could be altered by the experimental conditions.

Tissue-specific selection of stable reference genes for real-time PCR normalization in an obese rat model

Obesity is a complex pathology with interacting and confounding causes due to the environment, hormonal signaling patterns, and genetic predisposition. At present, the Zucker rat is an eligible genetic model for research on obesity and metabolic syndrome, allowing scrutiny of gene expression profiles. Real-time PCR is the benchmark method for measuring mRNA expressions, but the accuracy and reproducibility of its data greatly depend on appropriate normalization strategies. In the Zucker rat model, no specific reference genes have been identified in myocardium, kidney, and lung, the main organs involved in this syndrome. The aim of this study was to select among ten candidates (Actb, Gapdh, Polr2a, Ywhag, Rpl13a, Sdha, Ppia, Tbp, Hprt1 and Tfrc) a set of reference genes that can be used for the normalization of mRNA expression data obtained by real-time PCR in obese and lean Zucker rats both at fasting and during acute hyperglycemia. The most stable genes in the heart were Sdha, Tbp, and Hprt1; in kidney, Tbp, Actb, and Gapdh were chosen, while Actb, Ywhag, and Sdha were selected as the most stably expressed set for pulmonary tissue. The normalization strategy was used to analyze mRNA expression of tumor necrosis factor α, the main inflammatory mediator in obesity, whose variations were more significant when normalized with the appropriately selected reference genes. The findings obtained in this study underline the importance of having three stably expressed reference gene sets for use in the cardiac, renal, and pulmonary tissues of an experimental model of obese and hyperglycemic Zucker rats.

Comparison of NT-proCNP and CNP plasma levels in heart failure, diabetes and cirrhosis patients

C-type natriuretic peptide (CNP) plasma levels are extremely low and a pre-analytical phase is necessary to assay plasma CNP concentrations. Amino-terminal CNP (NT-proCNP) circulates at higher concentrations than CNP, allowing a direct assay and the use of smaller amounts of plasma. Aim of this study was to evaluate the analytical performance of a direct NT-proCNP assay and to measure its plasma levels in heart failure (CHF), diabetes and chirrosis patients. NT-proCNP and CNP were measured in 130 CHF, 19 patients with diabetes, 24 with hepatic cirrhosis and 73 controls. Plasma NT-proCNP was higher in all the clinical conditions studied (controls:45.5 ± 1.84 pg/ml, CHF:67.09 ± 7.36, diabetes:51.5 ± 5.75 cirrhosis:78.4 ± 19.9; p = 0.034, p = 0.04 controls vs. CHF and cirrhosis, respectively) and in CHF NT-proCNP concentration showed a significant increase as a function of clinical severity. By comparison of ROC curves, CNP assay resulted better associated with disease than NT-proCNP assay in all the different clinical conditions probably due to different release and clearance. The determination of NT-proCNP adds a piece of information to better understanding the molecular mechanisms at the basis of CNP action in different diseases. Due to its higher analytical feasibility, this determination could become widespread in clinical biochemistry laboratories and serve as a complementary marker of disease conditions.

Back to the heart: the protective role of adiponectin.

Cardiovascular disease (CVD) is the leading cause of death worldwide and the prevalence of obesity and diabetes are increasing. In obesity, adipose tissue increases the secretion of bioactive mediators (adipokines) that may represent a key mechanism linking obesity to CVD. Adiponectin, extensively studied in metabolic diseases, exerts anti-diabetic, anti-atherogenic and anti-inflammatory activities. Due to these positive actions, the role of adiponectin in cardiovascular protection has been evaluated in recent years. In particular, for its potential therapeutic benefits in humans, adiponectin has become the subject of intense preclinical research. In the cardiovascular context, understanding of the cellular and molecular mechanisms underlying the adiponectin system, throughout its secretion, regulation and signaling, is critical for designing new drugs that target adiponectin system molecules. This review focused on recent advances regarding molecular mechanisms related to protective effects of the adiponectin system on both cardiac and vascular compartments and its potential use as a target for therapeutic intervention of CVD.

Asymmetrical myocardial expression of natriuretic peptides in pacing-induced heart failure.

High-frequency pacing of the left ventricle (LV) free wall causes a dyssynchronous pattern of contraction that leads to progressive heart failure (HF) with pronounced differences in regional contractility. Aim of this study was to evaluate possible changes in brain natriuretic peptide (BNP) and C-type natriuretic peptide (CNP) mRNA expression in the anterior/anterior lateral region (pacing site, PS) as compared to the infero-septal region (opposite site, OS) and to explore possible association between the contractiling pattern and biomarker expression. Cardiac tissue was collected from minipigs with pacing-induced HF (n=8) and without (control, n=6). The samples were selectively harvested from the anterior left ventricular (LV) wall, PS, and from an area remote to the pacing-site, OS. BNP and CNP mRNA expression was evaluated by semi-quantitative polymerase chain reaction (PCR). A significant difference in BNP expression was found in the PS between HF animals and controls (BNP/GAPDH: 0.65+/-0.11 vs. 0.35+/-0.04, p=0.02), but not in the OS (BNP/GAPDH: 0.36+/-0.05, ns vs. controls). CNP expression was not different compared to controls, although higher levels were observed in the PS and in the OS with respect to the controls (CNP/GAPDH: controls 0.089+/-0.036, PS 0.289+/-0.23, OS 0.54+/-0.16). This finding was in tune with an increase of CNP tissue concentration (controls: 0.69+/-0.13; PS=1.56+/-0.19; OS=1.70+/-0.42 pg/mg protein; p=0.039 controls vs. OS). Higher BNP mRNA expression in the PS is consistent with a reduction in contractile function in this region, while higher CNP mRNA expression in the OS suggests the presence of concomitant endothelial dysfunction in the remote region.

IL-33/ST2 Pathway and Classical Cytokines in End-Stage Heart Failure Patients Submitted to Left Ventricular Assist Device Support: A Paradoxic Role for Inflammatory Mediators?

Background. Inflammation is a critical process contributing to heart failure (HF). We hypothesized that IL-33/ST2 pathway, a new mechanism regulated during cardiac stress, may be involved in the functional worsening of end-stage HF patients, candidates for left ventricular assist device (LVAD) implantation, and potentially responsible for their outcome. Methods. IL-33, ST2, and conventional cytokines (IL-6, IL-8, and TNF-α) were determined in cardiac biopsies and plasma of 22 patients submitted to LVAD implantation (pre-LVAD) and compared with (1) control stable chronic HF patients on medical therapy at the moment of heart transplantation without prior circulatory support (HT); (2) patients supported by LVAD at the moment of LVAD weaning (post-LVAD). Results. Cardiac expression of ST2/IL-33 and cytokines was lower in the pre-LVAD than in the HT group. LVAD determined an increase of inflammatory mediators comparable to levels of the HT group. Only ST2 correlated with outcome indices after LVAD implantation. Conclusions. IL-33/ST2 and traditional cytokines were involved in decline of cardiac function of ESHF patients as well as in hemodynamic recovery induced by LVAD. IL-33/ST2 pathway was also associated to severity of clinical course. Thus, a better understanding of inflammation is the key to achieving more favorable outcome by new specific therapies.

Impact of Obesity on the Expression Profile of Natriuretic Peptide System in a Rat Experimental Model

Natriuretic peptides (NPs) play an important role in obesity and aim of this study was to evaluate, in cardiac tissue of obese Zucker rats (O, n = 29) their transcriptomic profile compared to controls (CO, n = 24) by Real-Time PCR study; CNP protein expression was evaluated by immunostaining and immunometric tests. Myocardial histology was performed, confirming no alteration of organ structure. While ANP and BNP are cardiac peptides, CNP is mainly an endothelial hormone; thus its expression, as well as that of NPR-B and NPR-C, was also evaluated in kidney and lung of an animal subgroup (n = 20). In heart, lower BNP mRNA levels in O vs CO (p = 0.02) as well as ANP and CNP (p = ns), were detected. NPR-B/NPR-A mRNA was similar in O and CO, while NPR-C was numerically lower (p = ns) in O than in CO. In kidney, CNP/NPR-B/NPR-C mRNA was similar in O and CO, while in lung CNP/NPR-C expression decreased and NPR-B increased (p = ns) in O vs CO. Subdividing into fasting and hyperglycemic rats, the pattern of mRNA expression for each gene analyzed remained unchanged. The trend observed in heart, kidney and lung for CNP protein concentrations and immunohistochemistry reflected the mRNA expression. TNF-α and IL-6 mRNA were measured in each tissue and no significant genotype effect was detected in any tissue. The main NP variations were observed at the cardiac level, suggesting a reduced release by cardiac cells. The understanding of mechanisms involved in the modulation of the NP system in obesity could be a useful starting point for future clinical study devoted to identifying new obesity treatment strategies.

High concentration of C-type natriuretic peptide promotes VEGF-dependent vasculogenesis in the remodeled region of infarcted swine heart with preserved left ventricular ejection fraction.

BACKGROUND Vasculogenesis is a hallmark of myocardial restoration. Post-ischemic late remodeling is associated with pathology and function worsening. At the same time, neo-vasculogenesis helps function improving and requires the release of vascular endothelial growth factor type A (VEGF-A). The vasculogenic role of C-type natriuretic peptide (CNP), a cardiac paracrine hormone, is unknown in infarcted hearts with preserved left ventricular (LV) ejection fraction (EF). We explored whether myocardial VEGF-dependent vasculogenesis is affected by CNP. METHODS AND RESULTS To this end, infarcted swine hearts were investigated by magnetic resonance imaging (MRI), histological and molecular assays. At the fourth week, MRI showed that transmural myocardial infarction (MI) affected approximately 13% of the LV wall mass without impairing global function (LVEF>50%, n=9). Increased fibrosis, metalloproteases and capillary density were localized to the infarct border zone (BZ), and were associated with increased expression of CNP (p=0.03 vs. remote zone (RZ)), VEGF-A (p<0.001 vs. RZ), BNP, a marker of myocardial dysfunction (p<0.01 vs. RZ) and the endothelial marker, factor VIII-related antigen (p<0.01 vs. RZ). In vitro, CNP 1000 nM promoted VEGF-dependent vasculogenesis without affecting the cell growth and survival, although CNP 100 nM or a high concentration of VEGF-A halted vascular growth. CONCLUSIONS CNP expression is locally increased in infarct remodeled myocardium in the presence of dense capillary network. The vasculogenic response requires the co-exposure to high concentration of CNP and VEGF-A. Our data will be helpful to develop combined myocardial delivery of CNP and VEGF-A genes in order to reverse the remodeling process.

Sequencing and cardiac expression of natriuretic peptide receptors A and C in normal and heart failure pigs.

Pharmacological treatments able to activate natriuretic receptors (NPRs) and inhibit cardiac remodelling in heart failure (HF) patients, are currently under investigation. To better understand the therapeutic potential of the NPRs activation is necessary to dispose of experimental models devoid of confounding effects. The pig constitutes an animal model largely used but its genome is not completely sequenced. Aims of this study were to sequence NPR-A and NPR-C in Susscrofa and to evaluate ANP, BNP and NPRs mRNA expression in cardiac tissue of normal and HF minipigs in order to have a starting point for future studies devoted to check new potential drugs. Cardiac tissue was collected from adult male minipigs without (n=4) and with HF (n=5). Pig NPR-A (179bp) and NPR-C (203bp) mRNA were partially sequenced (GenBank n.: FJ518622, FJ518621). Compared to control, ANP and BNP gene expression resulted higher in all the cardiac chambers of HF heart. This increase is associated to a down-regulation of NPR-A and an up-regulation of NPR-C in HF. These sequences will provide a new tool to investigate the role of natriuretic peptides and of their receptors under physiological and pathological conditions and their response to therapeutic interventions.