Role of chromogranin A-derived fragments after resection of nonfunctioning pancreatic neuroendocrine tumors.

PURPOSE: No single reliable biomarker is available for nonfunctioning pancreatic neuroendocrine tumors (NF-PanNETs). Vasostatin-1 (VS-1), the N-terminal fragment of chromogranin A (CgA), seems to be a more accurate biomarker compared to its precursor. Primary aim was to investigate the ability of VS-1, compared to total-CgA, to assess the effectiveness of surgical resection performed for NF-PanNETs. Secondary aim was to evaluate two additional CgA-derived fragments, pancreastatin (PST) and vasostatin-2 (VS-2), as possible biomarkers for NF-PanNETs. METHODS: Consecutive patients who underwent surgery for NF-PanNETs at San Raffaele Scientific Institute were included (n = 35). Plasma levels of CgA and CgA-derived fragments were measured by Enzyme-Linked ImmunoSorbent Assay (ELISA), preoperatively and postoperatively. RESULTS: Preoperative VS-1 was significantly higher compared to VS-1 measured on postoperative day 5 (POD5) (pre: 0.338 nM versus POD5: 0.147 nM, P < 0.001), whereas total-CgA significantly increased after surgery (pre: 1.123 nM versus POD5: 1.949 nM, P = 0.006). Overall, 24 patients showed ≥ 1 feature of tumor aggressiveness (T3-T4, nodal/distant metastases, Ki67 > 5%, microvascular/perineural invasion, necrosis). The median percentage decrease in VS-1 plasma levels was 63% (IQR 28-88%) among patients with aggressive tumors, compared to 13% (IQR 0-57%) in the remaining population (P = 0.033). No significant differences in terms of PST (P = 0.870) and VS-2 (P = 0.909) were observed between preoperative and postoperative time. CONCLUSION: VS-1 provides an early assessment of surgical efficacy in patients who undergo resection for NF-PanNETs, especially in those with aggressive neoplasms. Total-CgA, PST and VS-2 have no clinical utility in this setting.

Real-time cellular impedance monitoring and imaging of biological barriers in a dual-flow membrane bioreactor.

The generation of physiologically relevant in-vitro models of biological barriers can play a key role in understanding human diseases and in the development of more predictive methods for assessing toxicity and drug or nutrient absorption. Here, we present an advanced cell culture system able to mimic the dynamic environment of biological barriers while monitoring cell behaviour through real-time impedance measurements and imaging. It consists of a fluidic device with an apical and a basal flow compartment separated by a semi-permeable membrane. The main features of the device are the integration of sensing through transepithelial electrical impedance (TEEI) measurements and transparent windows for optical monitoring within a dual flow system. Caco-2 cells were cultured in the TEEI bioreactor under both flow and static conditions. Although no differences in the expression of peripheral actin and occludin were visible, the cells in dynamic conditions developed higher impedance values at low frequencies, indicative of a higher paracellular electrical impedance with respect to the static cultures. TEEI measurements at high frequency also enabled monitoring monolayer formation, which can be correlated with the observation of an RC behaviour in the impedance spectra. In particular, the cells subject to flow showed accelerated barrier formation and increased vitality with respect to the static controls, again highlighting the importance of dynamic conditions for epithelial cells.

Oxidative stress enhances and modulates protein S-nitrosation in smooth muscle cells exposed to S-nitrosoglutathione.

Among S-nitrosothiols showing reversible binding between NO and -SH group, S-nitrosoglutathione (GSNO) represents potential therapeutics to treat cardiovascular diseases (CVD) associated with reduced nitric oxide (NO) availability. It also induces S-nitrosation of proteins, responsible for the main endogenous storage form of NO. Although oxidative stress parallels CVD development, little is known on the ability of GSNO to restore NO supply and storage in vascular tissues under oxidative stress conditions. Aortic rat smooth muscle cells (SMC) were stressed in vitro with a free radical generator (2,2'-azobis(2-amidinopropane) dihydrochloride, AAPH). The cellular thiol redox status was reflected through levels of reduced glutathione and protein sulfhydryl (SH) groups. The ability of GSNO to deliver NO to SMC and to induce protein S-nitrosation (investigated via mass spectrometry, MS), as well as the implication of two redox enzymes involved in GSNO metabolism (activity of gamma-glutamyltransferase, GGT, and expression of protein disulfide isomerase, PDI) were evaluated. Oxidative stress decreased both intracellular glutathione and protein -SH groups (53% and 32% respectively) and caused a 3.5-fold decrease of GGT activity, while PDI expression at the plasma membrane was 1.7-fold increased without any effect on extracellular GSNO catabolism. Addition of GSNO (50 µM) increased protein -SH groups and protein S-nitrosation (50%). Mass spectrometry analysis revealed a higher number of S-nitrosated proteins under oxidative stress (83 proteins, vs 68 in basal conditions) including a higher number of cytoskeletal proteins (15, vs 9 in basal conditions) related with cell contraction, morphogenesis and movement. Furthermore, proteins belonging to additional protein classes (cell adhesion, transfer/carrier, and transporter proteins) were S-nitrosated under oxidative stress. In conclusion, higher levels of GSNO-dependent S-nitrosation of proteins from the cytoskeleton and the contractile machinery were identified under oxidative stress conditions. The findings may prompt the identification of suitable biomarkers for the appraisal of GSNO bioactivity in the CVD treatment.

Solid recovered fuel: An experiment on classification and potential applications.

The residual urban waste of Prato district (Italy) is characterized by a high calorific value that would make it suitable for direct combustion in waste-to-energy plants. Since the area of central Italy lacks this kind of plant, residual municipal waste is quite often allocated to mechanical treatment plants in order to recover recyclable materials (such as metals) and energy content, sending the dry fractions to waste-to-energy plants outside the region. With the previous Italian legislation concerning Refuse Derived Fuels, only the dry stream produced as output by the study case plant, considered in this study, could be allocated to energy recovery, while the other output flows were landfilled. The most recent Italian regulation, introduced a new classification for the fuel streams recovered from waste following the criteria of the European standard (EN 15359:2011), defining the Solid Recovered Fuel (SRF). In this framework, the aim of this study was to check whether the different streams produced as output by the study case plant could be classified as SRF. For this reason, a sampling and analysis campaign was carried out with the purpose of characterizing every single output stream that can be obtained from the study case mechanical treatment plant, when operating it in different ways. The results showed that all the output flows from the study case mechanical treatment plant were classified as SRF, although with a wide quality range. In particular, few streams, of rather poor quality, could be fed to waste-to-energy plants, compatibly with the plant feeding systems. Other streams, with very high quality, were suitable for non-dedicated facilities, such as cement plants or power plants, as a substitute for coal. The implementation of the new legislation has hence the potential for a significant reduction of landfilling, contributing to lowering the overall environmental impact by avoiding the direct impacts of landfilling and by exploiting the beneficial effects of energy recovery from waste.

pGlu-serpinin protects the normotensive and hypertensive heart from ischemic injury.

Serpinin peptides derive from proteolytic cleavage of Chromogranin-A at C-terminus. Serpinin and the more potent pyroglutaminated-serpinin (pGlu-Serp) are positive cardiac ß-adrenergic-like modulators, acting through ß1-AR/AC/cAMP/PKA pathway. Because in some conditions this pathway and/or other pro-survival pathways, activated by other Chromogranin-A fragments, may cross-talk and may be protective, here we explored whether pGlu-Serp cardioprotects against ischemia/reperfusion injury under normotensive and hypertensive conditions. In the latter condition, cardioprotection is often blunted because of the limitations on pro-survival Reperfusion Injury Salvage Kinases (RISK) pathway activation. The effects of pGlu-Serp were evaluated on infarct size (IS) and cardiac function by using the isolated and Langendorff perfused heart of normotensive (Wistar Kyoto, WKY) and spontaneously hypertensive (SHR) rats exposed to ischemic pre-conditioning (PreC) and post-conditioning (PostC). In both WKY and SHR rat, pGlu-Serp induced mild cardioprotection in both PreC and PostC. pGlu-Serp administered at the reperfusion (Serp-PostC) significantly reduced IS, being more protective in SHR than in WKY. Conversely, left ventricular developed pressure (LVDevP) post-ischemic recovery was greater in WKY than in SHR. pGlu-Serp-PostC reduced contracture in both strains. Co-infusion with specific RISK inhibitors (PI3K/Akt, MitoKATP channels and PKC) blocked the pGlu-Serp-PostC protective effects. To show direct effect on cardiomyocytes, we pre-treated H9c2 cells with pGlu-Serp, which were thus protected against hypoxia/reoxygenation. These results suggest pGlu-Serp as a potential modulatory agent implicated in the protective processes that can limit infarct size and overcome the hypertension-induced failure of PostC.

Mechanostructure and composition of highly reproducible decellularized liver matrices.

Despite the increasing number of papers on decellularized scaffolds, there is little consensus on the optimum method of decellularizing biological tissue such that the micro-architecture and protein content of the matrix are conserved as far as possible. Focusing on the liver, the aim of this study was therefore to develop a method for the production of well-characterized and reproducible matrices that best preserves the structure and composition of the native extra cellular matrix (ECM). Given the importance of matrix stiffness in regulating cell response, the mechanical properties of the decellularized tissue were also considered. The testing and analysis framework is based on the characterization of decellularized and untreated samples in the same reproducible initial state (i.e., the equilibrium swollen state). Decellularized ECM (dECM) were characterized using biochemical, histological, mechanical and structural analyses to identify the best procedure to ensure complete cell removal while preserving most of the native ECM structure and composition. Using this method, sterile decellularized porcine ECM with highly conserved intra-lobular micro-structure and protein content were obtained in a consistent and reproducible manner using the equilibrium swollen state of tissue or matrix as a reference. A significant reduction in the compressive elastic modulus was observed for liver dECM with respect to native tissue, suggesting a re-examination of design parameters for ECM-mimicking scaffolds for engineering tissues in vitro.

Chromogranin A and the endothelial barrier function.

Chromogranin A (CgA) is an acidic glycoprotein belonging to a family of regulated secretory proteins stored in the dense core granules of many neuroendocrine cells and neurons. This protein is produced, in certain conditions also by cardiomyocytes, keratinocytes and granulocytes. Upon secretion CgA is released in the extracellular environment and then in circulation. Increased levels of circulating CgA have been detected in patients with cancer, heart failure, hypertension, atrophic gastritis, renal failure, giant cell artheritis, rheumatoid arthritis, sepsis and other inflammatory diseases. Endothelial cells, either those located in the close proximity of secretory cells or in distant tissues, may be exposed, therefore, to variable levels of CgA. In this review we discuss recent findings that implicate CgA and its fragments as a modulators of the physiology of endothelial cells in normal and in pathological conditions. In particular, we review data that suggest that CgA and its N-terminal fragment, called vasostatin-1, are important modulators of the endothelial barrier function and potent inhibitors of the endothelial cell activation caused by inflammatory and pro-angiogenic cytokines, with potential implications in angiogenesis, inflammation and cancer.

Influence of unsaturated carbonic acids on hemocompatibility and cytotoxicity of poly-vinylacetate based co-polymers.

The aim of this study was to investigate hemocompatibility and cytotoxicity properties of synthetic polymer coatings containing various unsaturated carbonic acids with vinylacetate. Co-polymers of vinylacetate and crotonic acid (CA), maleic acid (MA), and itaconic acid (IA) were made. The materials were characterized in terms of their adhesion to metal supports (titanium and stainless steel) as well as hemocompatibility (% hemolysis, wettability, erythrocyte aggregation, hemoglobin content, thrombocyte count and lipid peroxidation levels) and cytotoxicity (human endothelial cell activity in vitro and chromosome aberrations, bone marrow proliferation and cell ploidy in rats). Co-polymers of unsaturated carbonic acids with vinylacetate exhibited good hemocompatibility properties, as opposed to vinylacetate homopolymer for which substantial levels of hemolysis were observed. By coating the metal supports with co-polymers the cytotoxic effects associated with the bare metal samples were markedly reduced. MA samples showed excellent hemocompatibility and no cytotoxicity, yet they lacked good adhesion properties to metal substrate, probably due to high water content. CA samples, having the highest density of carboxylic groups among the samples under investigation, showed increased bone marrow proliferation activity and cell ploidy in rats, as compared to controls. The most promising results in the present study were obtained for the samples with IA, which showed good adhesion to metal substrates, good hemocompatibility and low cytotoxicity. Thus, co-polymers of vinylacetate and IA rich in carboxylic groups are promising materials for the design of novel drug-eluting stents.

Genetic inactivation of ApoJ/clusterin: effects on prostate tumourigenesis and metastatic spread.

ApoJ/Clusterin (CLU) is a heterodimeric protein localized in the nucleus, cytoplasm or secretory organelles and involved in cell survival and neoplastic transformation. Its function in human cancer is still highly controversial. In this study, we examined the prostate of mice in which CLU has been genetically inactivated. Surprisingly, we observed transformation of the prostate epithelium in the majority of CLU knockout mice. Either PIN (prostate intraepithelial neoplasia) or differentiated carcinoma was observed in 100 and 87% of mice with homozygous or heterozygous deletion of CLU, respectively. Crossing CLU knockout with TRAMP (prostate cancer prone) mice results in a strong enhancement of metastatic spread. Finally, CLU depletion causes tumourigenesis in female TRAMP mice, which are normally cancer free. Mechanistically, deletion of CLU induces activation of nuclear factor-kB, a potentially oncogenic transcription factor important for the proliferation and survival of prostate cells.

Acute liver failure: managing coagulopathy and the bleeding diathesis.

Acute liver failure (ALF) is defined as a severe, sudden liver dysfunction that induces encephalopathy and coagulopathy (prothrombin time [PT/INR] > 1.5) within 26 weeks of the onset of symptoms (usually jaundice) in patients without previous liver disease. Quantitative and qualitative platelet dysfunction, reduced synthesis of clotting factors, increased consumption of factors (mainly II, V, VII, X), reduced clearance of both activated factors, and/or factor inhibitor complexes are among the most important proposed pathogenetic factors. A possible role might be also played by the diminished degradation of anticoagulants. Plasminogen activator inhibitor 1 (PAI-1) is increased, shifting the balance toward hypofibrinolysis, despite the elevated levels of tissue plasminogen activator (tPA). Although changes in coagulation parameters provide crucial information for the management of the patient with ALF, the optimal management of the hemostatic defects is far from being defined. Because spontaneous bleeding occurs rarely during ALF, measures to improve the bleeding diathesis (fresh frozen plasma, cryoprecipitate, platelet transfusion) are recommended only in patients with clinically significant bleeding or before placement of invasive devices. Antifibrinolytic drugs are used in some cases, but often empirically. The role of rFVIIa, even if promising, is still under debate.

The endocrine role for chromogranin A: a prohormone for peptides with regulatory properties.

Chromogranin A (CgA) belongs to the granin family of uniquely acidic secretory proteins co-stored and co-secreted with other hormones and peptides in elements of the diffuse neuroendocrine system. The granins arise from different genes and are characterized by numerous sites for post-translational cleavage into shorter peptides with postulated regulatory properties. This review is directed towards endocrine aspects of CgA and its biologically active peptides. There is ample evidence from in vitro studies of distinct effects and targets for three CgA-derived peptides, vasostatin-I, pancreastatin and catestatin. Endocrine regulations are indicated from in vivo studies, consistent with the postulated prohormone function of CgA for peptides with regulatory properties. Most of the effects fit into patterns of direct or indirect, inhibitory modulations of major functions, implicating CgA peptides in regulation of calcium and glucose metabolism, cardiovascular functions, gastrointestinal motility and nociception, tissue repair, inflammatory responses and as host defense peptides in the first phase of microbial invasions.

Perioperative fluid management in kidney transplantation: is volume overload still mandatory for graft function?

Kidney transplantation is now recognized as the treatment of choice for patients with chronic renal failure. Despite the extension of indications to patients suffering severe hypertension, ischemic heart disease, and chronic heart failure, the worldwide results are superb. However, perioperative cardiac complications occur in 6% to 10% of transplanted patients. Aggressive intraoperative volume expansion is still recommended to maximize graft functional recovery (up to 30 mL/kg/h, central venous pressure [CVP] > 15 mm Hg), but patients with preexistent cardiac disease or poor myocardial function are exposed to the risk of fluid overload, acute respiratory failure, and prolonged ventilation. Among the last 90 cases performed at our institution, good functional recovery of the graft was present in 94% of the patients within 2 weeks, despite a much more conservative intraoperative hydration policy (crystalloids 2400 +/- 1000 mL, 15 mL/kg/h, CVP 7-9 mm Hg). Graft failure which occurred in 5 patients was significantly correlated only with donor age, while perioperative cardiovascular complications had been present in 9 cases (10%) who were coronary artery disease patients (55%). Age above 50 years was the only significant risk factor. Supranormal volume loading is probably not always warranted in kidney transplantation.

Effects of the tumor vasculature targeting agent NGR-TNF on the tumor microenvironment in murine lymphomas.

TNF-alpha may improve drug delivery to tumors by alteration of vascular permeability. However, toxicity precludes its systemic administration in patients. NGR-TNF comprises TNF coupled to the peptide CNGRC, which is a ligand for CD13. CD13 is expressed on tumor vasculature. Therefore, to assess the efficacy of NGR-TNF its biological effect on tumor vasculature should be measured rather than its effect on tumor growth. The aim of this study was to assess the effects of a low dose of NGR-TNF (5 ng/kg) on vascular permeability, tumor hypoxia, perfusion and proliferation in lymphoma bearing mice. MRI measurements with blood pool contrast agent showed an increased leakage of the contrast agent from the vasculature in NGR-TNF treated tumors compared with controls (p < 0.05), suggesting NGR-TNF-induced vascular permeability. Immunohistochemical analysis two hours after NGR-TNF treatment showed a decrease in tumor hypoxia (p < 0.1) and an increase in labeling index of the S-phase marker bromodeoxyuridine (p < 0.1), possibly due to an increase in tumor blood flow after NGR-TNF treatment. Although a decrease in tumor hypoxia and an increase in labeling index could have lead to increased tumor growth, in this experiment after one day a decrease in tumor volume was measured. Possibly, the effects on tumor hypoxia and proliferation two hours after treatment are transient and overruled by other, more longlasting effects. For example, the observed increase in vascular permeability may lead to haemoconcentration and increased interstitial pressure, ultimately resulting in an reduction of tumor blood flow and thus a decrease in tumor growth. A beneficial effect of NGR-TNF in combination with other therapeutical agents may therefore critically depend on the sequence and timing of the regimens. Currently, NGR-TNF is being tested in clinical studies.

Regulation of endothelial cell shape and barrier function by chromogranin A.

We have found that chromogranin A (CgA), a protein released in circulation by neuroendocrine cells and neurons, prevents the vascular leakage induced by tumor necrosis factor (TNF) in a mouse model. Studies of the mechanism of action showed that CgA and its NH(2)-terminal fragments inhibit TNF-induced vascular permeability by preventing endothelial cytoskeleton rearrangements. We propose that neuronal/endocrine secretion of CgA could contribute to the regulation of endothelial barrier function and the protection of vessels against plasma leakage in inflammatory diseases.

Vasostatins exert negative inotropism in the working heart of the frog.

An in vitro isolated working frog heart (Rana esculenta) was used to study the effects of exogenous CGA(1-76) (vasostatin 1), CGA(1-113) (vasostatin 2), and the synthetic CGA(7-57) on cardiac performance. Under basal cardiac conditions, the dose-response curves of the three peptides from 10(-8) to 10(-7) M showed a significant calcium-dependent negative inotropism that involved neither the endocardial endothelium nor the adrenergic and muscarinic receptors. In addition, the CgA fragments clearly counteracted the typical positive inotropism of isoprenaline (10(-<9) M). Taken together, these results provide the first evidence for a cardio-suppressive role for the vasostatins.

Chromogranin A in heart failure; a novel neurohumoral factor and a predictor for mortality.

BACKGROUND: In chronic heart failure, several hormonal systems are activated with diagnostic and prognostic implications. We tested the hypotheses that serum Chromogranin-A (CgA) -- a 49 kDa acid protein present in the secretor granules of neuroendocrine cells -- is increased in chronic heart failure and that CgA levels are a predictive factor for mortality. METHOD AND RESULTS: In 160 patients with chronic heart failure, we measured serum CgA and other neuroendocrine hormones. The results showed that CgA is increased in chronic heart failure and the increase is related to the clinical severity of the syndrome: CgA levels in New York Heart Failure (NYHA) class II (median 146.9 ng x ml(-1), inter-quartiles 108.3-265.5) were significantly higher (P<0.05) than in class I (median 109.7 ng x ml(-1), inter-quartiles 96.7-137.6), and significantly lower (P<0.05) than in class III (median 279.0 ng x ml(-1), inter-quartiles 203.6-516.1). Class IV patients showed the highest serum levels of CgA (median 545.0 ng. ml(-1), inter-quartiles 231.8-1068.3), being statistically significantly different from class III patients (P<0.001). The association between survival and some recognized variables of prognostic significance, including CgA was also studied. The results showed that ejection fraction, noradrenaline, atrial natriuretic peptide, NYHA class and CgA were significant univariate prognosticators; however, in the multivariate analysis by the Cox proportional-hazard model, CgA and NYHA class were the only independent predictive factors for mortality (P<0.005, RR=1.22, 95% CI=1.06-1.41 and P=0.04, RR=1.58, 95% CI=1.02-2.46, respectively). CONCLUSIONS: CgA is a pro-hormone, precursor of several active fragments likely to exert biological effects in chronic heart failure. CgA serum levels are increased in patients with chronic heart failure and are a predictive factor for mortality.