Maria Angela D'Amico

Biological function and clinical relevance of chromogranin A and derived peptides

Chromogranin A (CgA (CHGA)) is the major soluble protein co-stored and co-released with catecholamines and can function as a pro-hormone by giving rise to several bioactive peptides. This review summarizes the physiological functions, the pathogenic implications, and the recent use of these molecules as biomarkers in several pathological conditions. A thorough literature review of the electronic healthcare databases MEDLINE, from January 1985 to September 2013, was conducted to identify articles and studies concerned with CgA and its processing. The search strategies utilized keywords such as chromogranin A, vasostatins 1 and 2, chromofungin, chromacin, pancreastatin, catestatin, WE14, chromostatin, GE25, parastatin, and serpinin and was supplemented by the screening of references from included papers and review articles. A total of 209 English-language, peer-reviewed original articles or reviews were examined. The analysis of the retrospective literature suggested that CgA and its several bioactive fragments exert a broad spectrum of regulatory activities by influencing the endocrine, the cardiovascular, and the immune systems and by affecting the glucose or calcium homeostasis. As some peptides exert similar effects, but others elicit opposite responses, the regulation of the CgA processing is critical to maintain homeostasis, whereas an unbalanced production of peptides that exert opposing effects can have a pathogenic role in several diseases. These clinical implications entail that CgA and its derived peptides are now used as diagnostic and prognostic markers or to monitor the response to pharmacological intervention not only in endocrine tumors, but also in cardiovascular, inflammatory, and neuropsychiatric diseases.

Functional mitral regurgitation: from normal to pathological anatomy of mitral valve.

Mitral valve (MV) is composed of several structures working in synchrony to open during diastole and close in systole within the high-pressure systemic environment. Its morphological features ensure a normal leaflet closure that prevents regurgitation of blood back into the left atrium causing loss of ventricular pressure and forward flow. The complex interactions of the normal MV are reliant on each component playing a complete role for the efficient working of the valve. In this review we firstly discuss the overall MV structure in terms of a complex make up of the annulus, the leaflets, their tendinous cords, and the supporting papillary muscles, and then the anatomical changes of each MV components due to left ventricular geometry and function alterations, underlying functional mitral regurgitation.

Human second trimester amniotic fluid cells are able to create embryoid body-like structures in vitro and to show typical expression profiles of embryonic and primordial germ cells.

Human amniotic fluid-derived stem cells (AFSCs) represent a novel class of broadly multipotent stem cells sharing characteristics of both embryonic and adult stem cells. However, both the origin of these cells and their actual properties in terms of pluripotent differentiation potential are still debated. In order to verify the presence of features of pluripotency in human second trimester AFSCs, we have investigated the ability of these cells to form in vitro three-dimensional aggregates, known as embryoid bodies (EBs), and to express specific genes of embryonic stem cells (ESCs) and primordial germ cells (PGCs). EBs were obtained after 5 days of AFSC culture in suspension and showed positivity for alkaline phosphatase (AP) staining and for specific markers of pluripotency (OCT4 and SOX2). Moreover, EB-derived cells showed the expression of specific transcripts of the three germ layers. RT-PCR analysis, carried out at different culture times (second, third, fourth, fifth, and eighth passages), revealed the presence of specific markers of ESCs (such as FGF4 and DAPPA4), as well as of markers typical of PGCs and, in particular, genes involved in early stages of germ cell development (Fragilis, Stella, Vasa, c-Kit, Rnf17). Finally, the expression of genes related to the control of DNA methylation (DNMT3A, DNMT3b1, DNMT1, DNMT3L, MBD1, MBD2, MBD3, MDB4, MeCP2), as well as the lack of inactivation of the X-chromosome in female samples, was also demonstrated. Taken together, these data provide further evidence for the presence of common features among human AFSCs, PGCs, and ESCs.

Effects of ACE I/D polymorphism and aerobic training on the immune-endocrine network and cardiovascular parameters of postmenopausal women

CONTEXT Estrogen deficiency, systemic low-grade inflammation, and reduction of adrenal gland function have central roles in noncommunicable chronic disease (NCD) development. With angiotensin-converting enzyme (ACE) insertion/deletion (I/D) polymorphism, the deletion variant (DD) is related to higher levels of circulating angiotensin II than I allele carriers (II/ID), which might interact with all of these molecular pathways to increase NCDs risk. On the other hand, physical exercise counteracts the occurrence of NCDs, potentially acting on the same pathways. OBJECTIVES The aim of the study was to investigate the effects of walking training on adrenal steroid and cytokine levels and on cardiovascular parameters in postmenopausal women with ACE I/D genotypes. METHODS Thirty-six (DD = 15, II/ID = 21) sedentary postmenopausal women (mean age, 56 ± 4 y) participated in a 13-week program of walking training at moderate intensity. Heart rate, blood pressure, double product, TNF-α, dehydroepiandrosterone sulfate (DHEA-S), and cortisol were evaluated before and after the intervention program. RESULTS Before walking training, the ACE DD genotype showed significantly higher TNF-α (P = .007) and lower DHEA-S concentrations (P = .022) than the ACE II/ID individuals. After walking training, both subgroups significantly decreased TNF-α plasma levels and cortisol/DHEA-S ratio (P = .001 and P = .016, respectively) and significantly increased DHEA-S levels (P < .001). Moreover, all the cardiovascular parameters were significantly reduced in the ACE DD participants (P ≤ .05), whereas the ACE I-allele carriers showed a decrease in heart rate (P ≤ .05) and the double product (P ≤ .05). CONCLUSION ACE I/D polymorphism is linked to different adrenal steroid and cytokine levels, and ACE I-allele carriers show a better adrenal activity and systemic inflammatory profile. The introduction of walking training positively influences the menopause immune-neuroendocrine changes, independent of ACE I/D genotype.

IL‐6 Activates PI3K and PKCζ Signaling and Determines Cardiac Differentiation in Rat Embryonic H9c2 Cells

Introduction: IL‐6 influences several biological processes, including cardiac stem cell and cardiomyocyte physiology. Although JAK‐STAT3 activation is the defining feature of IL‐6 signaling, signaling molecules such as PI3K, PKCs, and ERK1/2 are also activated and elicit different responses. Moreover, most studies on the specific role of these signaling molecules focus on the adult heart, and few studies are available on the biological effects evoked by IL‐6 in embryonic cardiomyocytes. Aim: The aim of this study was to clarify the biological response of embryonic heart derived cells to IL‐6 by analyzing the morphological modifications and the signaling cascades evoked by the cytokine in H9c2 cells. Results: IL‐6 stimulation determined the terminal differentiation of H9c2 cells, as evidenced by the increased expression of cardiac transcription factors (NKX2.5 and GATA4), structural proteins (α‐myosin heavy chain and cardiac Troponin T) and the gap junction protein Connexin 43. This process was mediated by the rapid modulation of PI3K, Akt, PTEN, and PKCζ phosphorylation levels. PI3K recruitment was an upstream event in the signaling cascade and when PI3K was inhibited, IL‐6 failed to modify PKCζ, PTEN, and Akt phosphorylation. Blocking PKCζ activity affected only PTEN and Akt. Finally, the overexpression of a constitutively active form of PKCζ in H9c2 cells largely mimicked the morphological and molecular effects evoked by IL‐6. Conclusions: This study demonstrated that IL‐6 induces the cardiac differentiation of H9c2 embryonic cells though a signaling cascade that involves PI3K, PTEN, and PKCζ activities. J. Cell. Physiol. 231: 576–586, 2016.

NAD(P)H oxidase p22(phox) polymorphism and cardiovascular function in amateur runners.

NAD(P)H system represents the major source of superoxide production at cardiovascular (CV) level. It has several genetic variants: in particular, the C242T polymorphism of its p22phox subunit is associated with a different oxidase activity, being the T allele related to a lower superoxide production. Although several authors investigated the protective effect of T allele in CV diseases, only few data are available on its functional role in physiological conditions. The aim of our study was to investigate the relationship between the p22phox C242T polymorphism and CV function in amateur runners.

Novel evidence of ghrelin and growth hormone segretagogue receptor expression by human ocular tissues

AIM OF THE STUDY The gastrointestinal peptide hormone ghrelin (Ghr) was discovered in 1999 as the endogenous ligand for the growth hormone secretagogue receptor (GHSR-1a). It is a pleiotropic peptide that modulates a wide spectrum of biological activities, such as growth hormone (GH) release, feeding stimulation, adiposity and cardiovascular actions. The presence of Ghr mRNA in the iris and ciliary body (CB) epithelium was recently demonstrated in animal models, where a possible myorelaxing effect on the iris muscles has been suggested. Based on these observations, the aim of our study was to investigate the Ghr and GHSR-1a expression and localization in the normal human eye. MATERIAL Five different ciliary body/iris samples from normal eyes were subjected to Western blot analysis. Immunohistochemical detection was performed on three enucleated eyes. Twenty aqueous humor (AqH) samples obtained from patients submitted to cataract surgery were analyzed with an ELISA for the presence of Ghr. RESULTS Ghr and GHSR-1a were co-expressed by the pigmented epithelium (PE) of the CB, by the retinal pigmented epithelium (RPE) and by the anterior limiting layer (ALL) of the iris. No reaction was detected at the subepithelial level in the ciliary or pupillae smooth muscle cells. The AqH samples were positive for the presence of Ghr. CONCLUSION This study provides the first evidence that Ghr and GHSR-1a are expressed in the human eye by specific cells. The understanding of the functional role of Ghr at the human eye level needs more efforts and investigation, but a hypothetical action on the GH retinal synthesis and/or on the circadian clock system could be suggested.

Psychophysiological responses of junior orienteers under competitive pressure

The purpose of the study was to examine psychobiosocial states, cognitive functions, endocrine responses (i.e., salivary cortisol and chromogranin A), and performance under competitive pressure in orienteering athletes. The study was grounded in the individual zones of optimal functioning (IZOF) and biopsychosocial models. Fourteen junior orienteering athletes (7 girls and 7 boys), ranging in age from 15 to 20 years (M = 16.93, SD = 1.77) took part in a two-day competitive event. To enhance competitive pressure, emphasis was placed on the importance of the competition and race outcome. Psychophysiological and performance data were collected at several points before, during, and after the races. Results showed that an increase in cortisol levels was associated with competitive pressure and reflected in higher perceived exertion (day 1, r = .32; day 2, r = .46), higher intensity of dysfunctional states (day 1, r = .59; day 2, r = .55), lower intensity of functional states (day 1, r = -.36; day 2, r = -.33), and decay in memory (day 1, r = -.27; day 2, r = -.35), visual attention (day 1, r = -.56; day 2, r = -.35), and attention/mental flexibility (day 1, r = .16; day 2, r = .26) tasks. The second day we observed better performance times, lower intensity of dysfunctional states, lower cortisol levels, improved visual attention and attention/mental flexibility (p < .050). Across the two competition days, chromogranin A levels were higher (p < .050) on the most difficult loops of the race in terms of both physical and psychological demands. Findings suggest emotional, cognitive, psychophysiological, and performance variables to be related and to jointly change across different levels of cognitive and physical load. Overall results are discussed in light of the IZOF and biopsychosocial models. The procedure adopted in the study also supports the feasibility of including additional cognitive load for possible practical applications.

Amniotic Fluid Stem Cells cardiomyogenic potential: a preliminary study

The characterization of Amniotic Fluid-derived multipotent Stem Cells (AFSCs) open new paths in stem cell research. hAFSCs have characteristics intermediate between pluripotent embryonic- (ESCs) and lineage-restricted adult stem cells, and are non-tumorigenic and low immunogenic. Moreover, they are obtained without destroying human embryos, so that most of the ethical and social controversy could be prevented. We previously observed that human AFSCs express some genes specific of ESCs and primordial germ cells. We also shown hAFSCs ability to form in vitro three-dimensional aggregates of cells known as embryoid bodies (EBs), that express three germ layer markers. Recent studies reported the ability of hAFSCs to differentiate in vitro into adipocytes and osteocytes. Aim of our study was to analyse the cardiomyogenic potential of hAFSCs. EBs were obtained by modified hanging drops protocol from hAFSCs coltured in presence of ascorbic acid and 5-aza-2’-deoxycytidine (differentiation medium: DM). RT-PCR and Western Blotting analysis conducted on AFSCs and EBs cells evidenced the gene and protein expression of the transcriptor factor Nkx2.5, the earliest marker of heart precursor cells. Immunofluorescence (IF) analysis performed on EBs after 10 days in DM evidenced the cytoplasmic presence of α-myosin heavy chain (α-MHC) organized in parallel, oriented filamets. Microscopical analysis evidenced beating cells mainly at the periphery of the EB. In conclusion, our results evidenced that hAFSCs cultured in permissive conditions give rise to EB able to terminally differentiate in cardiomyocytes.

The phosphoinositide 3-kinase role in Il-6 induction to differentiation of rat embryonic cardiomyocytes

Purpose . Cardiomyocytes express IL-6 and its signal transducer, 130-kDa glycoprotein (gp130) that influence cell growth, apoptosis, differentiation and survival by an autocrine pathway. High levels of circulating IL-6 have been reported in patients with congestive heart failure (HF) and after myocardial infarction and IL-6-gp130-signaling participates in HF prevention and compensatory hypertrophy, influencing remodeling processes and inducing Protein kinase C (PKC)-dependent apoptosis. Recent studies demonstrated the presence of precursor cells able to differentiate into cardiomyocytes in the adult heart through signaling pathways similar to those in the embryonic heart thus participating to the physiologic repair of the injured heart. In this line, the study of the embryonic signaling may represent an important cue to understand the molecular bases of the regenerative capacity myocardial progenitors. Aim of this study was to investigate the signal transduction pathways evoked by IL-6 treatment on cardiomyoblasts and its possible differentiating effects. Methods . H9c2 cells cultured in medium supplemented with 1% FBS (differentiation-promoting medium) in presence of IL-6 (10 ng/ml) up to three days. Results . Western Blot and Immunofluorence analysis demonstrated that Il-6 after 3 days of treatment induced a marked increase of the expression of α-myosin heavy chain (α-MHC; terminal cardiac differentiation marker) together with a microfilament reorganization and morphological modifications, that included mono-nucleated cells elongation and fusion into multinucleated tubes. This process was accompanied by an evident nuclear translocation of Nkx2.5 (early myocardial development transcription factor), and by a sub-cellular redistribution of gp130. Results suggest that the biological effects evoked by IL6 were at least partially mediated by PI3K through Akt and PKCzeta pathways modulation, as confirmed by the varied phosphorylated PI3K level observed after 30 min of IL-6 treatment and PI3K induction of downstream PKCzeta phosphorilation, but not PKCalpha and PKCdelta, whereas Akt de-phosphorylation was detected. Conclusions . Our observations evidenced that PI3K is a key regulator of IL-6- induction of embryonic cardiomyocytes differentiation by PKCzeta and Akt-signalling pathways. These data suggest a possible role of IL-6 dependent pathways in regenerative capacity of myocardial cells following injuries.