S. Del Ry

Bleomycin in the setting of lung fibrosis induction: From biological mechanisms to counteractions.

Bleomycin (BLM) is a drug used to treat different types of neoplasms. BLMs most severe adverse effect is lung toxicity, which induces remodeling of lung architecture and loss of pulmonary function, rapidly leading to death. While its clinical role as an anticancer agent is limited, its use in experimental settings is widespread since BLM is one of the most widely used drugs for inducing lung fibrosis in animals, due to its ability to provoke a histologic lung pattern similar to that described in patients undergoing chemotherapy. This pattern is characterized by patchy parenchymal inflammation, epithelial cell injury with reactive hyperplasia, epithelial-mesenchymal transition, activation and differentiation of fibroblasts to myofibroblasts, basement membrane and alveolar epithelium injuries. Several studies have demonstrated that BLM damage is mediated by DNA strand scission producing single- or double-strand breaks that lead to increased production of free radicals. Up to now, the mechanisms involved in the development of pulmonary fibrosis have not been fully understood; several studies have analyzed various potential biological molecular factors, such as transforming growth factor beta 1, tumor necrosis factor alpha, components of the extracellular matrix, chaperones, interleukins and chemokines. The aim of this paper is to review the specific characteristics of BLM-induced lung fibrosis in different animal models and to summarize modalities and timing of in vivo drug administration. Understanding the mechanisms of BLM-induced lung fibrosis and of commonly used therapies for counteracting fibrosis provides an opportunity for translating potential molecular targets from animal models to the clinical arena.

Clinical relevance of cardiac natriuretic peptides measured by means of competitive and non-competitive immunoassay methods in patients with renal failure on chronic hemodialysis

Increased levels of cardiac natriuretic peptides in patients undergoing hemodialysis may be a marker of cardiomyopathy and in consequence may be suitable prognostic indicators for the risk of development of cardiac disease. We measured plasma levels of ANP, BNP, proANP1–98 and proBNP1–76-related peptides with some competitive and non-competitive immunoassay methods in patients with renal failure on chronic hemodialysis in order to compare the analytical performances of these methods and to evaluate the clinical usefulness of each assay for patients with chronic renal failure. ANP and BNP values significantly decreased after hemodialysis (on average, ANP by 36% and BNP by 16%); while all proANP and proBNP values tended to increase, but only proANP1–30 (by 14.4%) and Nt-proBNP (by 9.5%) significantly. Although significant correlations were found among all the circulating levels of cardiac peptides studied, N-terminal pro-peptides correlated better among themselves than with ANP and BNP; ANP was only slightly correlated with all the other peptides, the only exception being BNP. Only BNP levels significantly increased according to the degree of ventricular hypertrophy and/or ventricular function in patients with chronic renal failure. The ANP assay is preferable in physiological and clinical studies for the rapid changes in atrial pre-load. BNP would be more useful in the follow-up of cardiac complications in patients with end-stage renal disease on regular hemodialysis. The assays of N-terminal pro-ANP1–98 -and proBNP1-76-related peptides proved to be of limited use, because they were not able to detect acute changes in pre-load during hemodialysis and were less useful than BNP levels as markers of ventricular hypertrophy and/or functional cardiac impairment.

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.

Possibility of age regulation of the natriuretic peptide C-receptor in human platelets

Natriuretic peptide binding sites on platelets have been hypothesized to act as clearance receptors; however, there is no clear definition of the function of this receptor. The aim of the study was: 1) to characterize natriuretic peptide receptors in human platelets by original competition study; 2) to evaluate a possible age modulation of these binding sites, since a delayed clearance of ANP in the elderly has been observed. The binding of 125I-ANP to intact platelets was completely inhibited by h-ANP, h-BNP, h-CNP and c-ANP, the selective ligand of the clearance receptor. IC50 values were 0.089±0.029, 0.703±0.104 and 1.19±0.13, 3.84±0.04 nmol/l, mean±SE, respectively (p<0.001 for IC50 value of h-ANP compared to the other natriuretic peptides). This observation on the receptor selectivity of natriuretic peptides in human platelets provides new evidence for the presence of the clearance receptor on platelets. In control subjects the Kd was 34.6±4.0 pmol/l and Bmax 13.6±0.92 fmol/109 platelets (mean±SE), (no.=46, mean age 41.7±2.1 years). Bmax was significantly reduced in older subjects (no.=25, mean age 53.2±1.5 years) with respect to the younger group (no.=21, mean age 28.0±0.87 years): 11.4±1.1 vs 16.1±1.4 fmol/109 cells, p=0.0096, respectively; moreover, a significant inverse relationship between Bmax and the subject’s age was observed. This observation suggests a possible reduction of the natriuretic peptide clearance with aging, associated to a significant increase of plasma levels of natriuretic peptides.

C-type natriuretic peptide: A new cardiac mediator

Natriuretic peptides are endogenous hormones released by the heart in response to myocardial stretch and overload. While atrial and brain natriuretic peptides (ANP, BNP) were immediately considered cardiac hormones and their role was well-characterized and defined in predicting risk in cardiovascular disease, evidence indicating the role of C-type natriuretic peptide (CNP) in cardiovascular regulation was slow to emerge until about 8 years ago. Since then, considerable literature on CNP and the cardiovascular system has been published; the aim of this review is to examine current literature relating to CNP and cardiovascular disease, in particular its role in heart failure (HF) and myocardial infarction (MI). This review retraces the fundamental steps in research that led understanding the role of CNP in HF and MI; from increased CNP mRNA expression and plasmatic concentrations in humans and in animal models, to detection of CNP expression in cardiomyocytes, to its evaluation in human leukocytes. The traditional view of CNP as an endothelial peptide has been surpassed by the results of many studies published in recent years, and while its physiological role is still under investigation, information is now available regarding its contribution to cardiovascular function. Taken together, these observations suggest that CNP and its specific receptor, NPR-B, can play a very important role in regulating cardiac hypertrophy and remodeling, indicating NPR-B as a new potential drug target for the treatment of cardiovascular disease.Abstract Natriuretic peptides are endogenous hormones released by the heart in response to myocardial stretch and overload. While atrial and brain natriuretic peptides (ANP, BNP) were immediately considered cardiac hormones and their role was well-characterized and defined in predicting risk in cardiovascular disease, evidence indicating the role of C-type natriuretic peptide (CNP) in cardiovascular regulation was slow to emerge until about 8 years ago. Since then, considerable literature on CNP and the cardiovascular system has been published; the aim of this review is to examine current literature relating to CNP and cardiovascular disease, in particular its role in heart failure (HF) and myocardial infarction (MI). This review retraces the fundamental steps in research that led understanding the role of CNP in HF and MI; from increased CNP mRNA expression and plasmatic concentrations in humans and in animal models, to detection of CNP expression in cardiomyocytes, to its evaluation in human leukocytes. The traditional view of CNP as an endothelial peptide has been surpassed by the results of many studies published in recent years, and while its physiological role is still under investigation, information is now available regarding its contribution to cardiovascular function. Taken together, these observations suggest that CNP and its specific receptor, NPR-B, can play a very important role in regulating cardiac hypertrophy and remodeling, indicating NPR-B as a new potential drug target for the treatment of cardiovascular disease.

C-type natriuretic peptide and its relation to non-invasive indices of left ventricular function in patients with chronic heart failure.

C-type natriuretic peptide (CNP) significantly increases in chronic heart failure (CHF) patients as a function of clinical severity. Aim of this study was to evaluate in CHF patients the relationship between circulating CNP concentrations and echo-Doppler conventional indices of left ventricular (LV) function as well as less load independent parameters as dP/dt. LV ejection fraction (EF), left ventricular end-diastolic dimension (LVEDD) and LV dP/dt were evaluated together with plasma CNP levels in 38 patients with CHF and in 63 controls. CNP levels resulted significantly higher in CHF patients than in controls (7.19+/-0.59 pg/ml vs. 2.52+/-0.12 pg/ml, p<0.0001). A significant correlation between dP/dt and CNP levels (r=-0.61, p<0.0001) was observed. A good correlation with EF (r=-0.55, p<0.001) and a less significant relation with LVEDD (r=0.316, p<0.05) were also reported. When patients were divided according to dP/dt values a very significant difference in CNP levels was observed: Group I (<600, n=25) vs. Group II (>600, n=13): 8.46+/-0.69 and 4.75+/-0.75 pg/ml, respectively, p<0.001. This is the first study that reports a correlation between CNP and dP/dt in CHF patients, thus suggesting a possible role on cardiac contractility.

Circulating levels of cardiac natriuretic hormones measured in women during menstrual cycle

Alterations in fluid and electrolyte balance represent a common complaint by women during different stages of the menstrual cycle; however, conflicting results concerning the possible role of plasma Atrial Natriuretic Peptide (ANP) modifications during the menstrual cycle have been reported. This may be due to differences in assay methods or in the clinical protocol adopted. Moreover, possible variations in plasma Brain Natriuretic Peptide (BNP) levels during the menstrual cycle have not been studied. We measured the plasma levels of ANP and BNP by means of two highly sensitive and specific immunoradiometric assay (IRMA) methods in 19 normal women without premenstrual symptoms, in order to evaluate whether significant modifications of these hormones are present during the menstrual cycle. Because it is well-known that circulating levels of cardiac hormones show great variations in normal subjects due to their rapid plasma half lifes, blood samples were collected at 2.5-min intervals over a 15-min period on the 5th and 24th days of the cycle. The mean (±SD) values of ANP (follicular phase=15.1±8.7 pg/ml; luteal phase=14.8±9.5 pg/ml) and of BNP (follicular phase=13.0±15.0 pg/ml; luteal phase= 11.2±11.4 pg/ml) did not show significant variations during the menstrual cycle. Moreover, the variability of ANP values (CV=24.8±13.2%) was significantly higher (p=0.0318) than that of BNP values (CV=16.5±8.9%), and a significant correlation was found between the mean ANP and BNP values of the individual women studied (R=0.407, p=0.0437). The values of estradiol, progesterone, LH, FSH and prolactin did not correlate with the ANP or BNP values. In conclusion, our results indicate that circulating levels of cardiac hormones do not show any significant modifications during the menstrual cycle in healthy women.

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.

Plasma C-type natriuretic peptide levels in healthy children.

C-type natriuretic peptide (CNP) is assuming increasing importance in cardiovascular disease, and in adults its plasma levels are related to clinical and functional disease severity. Data are scarce regarding the reference values for CNP in infancy. Aim of this study was to assess the reference intervals for CNP in human healthy newborns and infants. Plasma CNP was measured in 121 healthy children divided into: 41 newborns (age 0-3 days), 24 newborns (4-30 days), 22 infants (1-12 months) and 32 children (1-12 years). A group of 32 healthy adult subjects (age 64 ± 1 years) was also studied. CNP was measured by a specific radioimmunoassay. Between- and within-assay variability resulted ≤ 30 and 20%, respectively and analytical sensitivity 0.77 ± 0.05 pg/tube. Plasma CNP resulted significantly higher in children than in adult subjects (13.6 ± 1.2 pg/ml vs. 7.4 ± 1.0 pg/ml, p=0.030). When the results were analyzed as a function of the age the reference intervals for plasma CNP resulted: 11.6 ± 2.1 pg/ml for newborns (0-3 days), 16.4 ± 3.7 pg/ml for newborns (4-30 days), 15.4 ± 2.7 pg/ml for infants (1-12 months), 13.6 ± 2.3 pg/ml for children (1-12 years) [p=0.01 newborns (4-30 days) vs. adults; p=0.03 infants (1-12 months) vs. adults]. CNP showed the highest concentrations after 12h of life with a peak between 4 and 5 days of life and with a progressive decline afterwards. According to these data at least five different reference intervals for CNP determinations should be used. These observations may be helpful for future clinical application of CNP in human children.

Gene expression of C-type natriuretic peptide and of its specific receptor NPR-B in human leukocytes of healthy and heart failure subjects

C-type natriuretic peptide (CNP), a member of the family of natriuretic peptides, is synthesized and secreted from monocytes and macrophages that resulted to be a source of CNP at inflammatory sites. This suggests that special attention should be focused on the possible role of CNP in the immune system, in addition to its effects on the cardiovascular system. The aim of this study was to evaluate the possibility of measuring the mRNA expression of CNP and NPR-B, its specific receptor, in human whole blood samples of healthy (N; n=7) and heart failure (HF; n=7) subjects by Real-Time PCR (RT-PCR). Total RNA was extracted from leukocytes with QIAamp RNA Blood Kit and/or with PAXgene Blood RNA Kit. RT-PCR was performed and optimized for each primer. The experimental results were normalized with the three most stably expressed genes. CNP and NPR-B expression trend was similar in both fresh and frozen human whole blood. Significant higher levels of CNP and NPR-B mRNA expression were found in HF patients with respect to controls (CNP: N=1.23±0.33 vs. HF=6.54±2.09 p=0.027; NPR-B: N=0.85±0.23 vs. HF=5.31±1.98 p=0.04). A significant correlation between CNP and NPR-B (r=0.86, p<0.0001) was observed. Further studies are needed to clarify the pathophysiological properties of this peptide but the possibility to measure CNP and NPR-B mRNA expression in human leukocytes with a fast and easy procedure is a useful starting point for future investigation devoted to better understand the biomolecular processes associated to different diseases.