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BACKGROUND:Endothelial progenitor cells (EPCs) may be recruited from bone marrow to sustain tumor vascularisation and promote the metastatic switch. Understanding the molecular mechanisms driving EPC proliferation and tubulogenesis could outline novel targets for alternative anti-angiogenic treatments. Store-operated Ca(2+) entry (SOCE), which is activated by a depletion of the intracellular Ca(2+) pool, regulates the growth of human EPCs, where is mediated by the interaction between the endoplasmic reticulum Ca(2+)-sensor, Stim1, and the plasmalemmal Ca(2+) channel, Orai1. As oncogenesis may be associated to the capability of tumor cells to grow independently on Ca(2+) influx, it is important to assess whether SOCE regulates EPC-dependent angiogenesis also in tumor patients. METHODOLOGY/PRINCIPAL FINDINGS:The present study employed Ca(2+) imaging, recombinant sub-membranal and mitochondrial aequorin, real-time polymerase chain reaction, gene silencing techniques and western blot analysis to investigate the expression and the role of SOCE in EPCs isolated from peripheral blood of patients affected by renal cellular carcinoma (RCC; RCC-EPCs) as compared to control EPCs (N-EPCs). SOCE, activated by either pharmacological (i.e. cyclopiazonic acid) or physiological (i.e. ATP) stimulation, was significantly higher in RCC-EPCs and was selectively sensitive to BTP-2, and to the trivalent cations, La(3+) and Gd(3+). Furthermore, 2-APB enhanced thapsigargin-evoked SOCE at low concentrations, whereas higher doses caused SOCE inhibition. Conversely, the anti-angiogenic drug, carboxyamidotriazole (CAI), blocked both SOCE and the intracellular Ca(2+) release. SOCE was associated to the over-expression of Orai1, Stim1, and transient receptor potential channel 1 (TRPC1) at both mRNA and protein level The intracellular Ca(2+) buffer, BAPTA, BTP-2, and CAI inhibited RCC-EPC proliferation and tubulogenesis. The genetic suppression of Stim1, Orai1, and TRPC1 blocked CPA-evoked SOCE in RCC-EPCs. CONCLUSIONS:SOCE is remodelled in EPCs from RCC patients and stands out as a novel molecular target to interfere with RCC vascularisation due to its ability to control proliferation and tubulogenesis.

Endothelial progenitor cells (EPCs) have recently been employed in cell-based therapy (CBT) to promote neovascularization and regeneration of ischemic organs, such as heart and limbs. Furthermore, EPCs may be recruited from bone marrow by growing tumors to drive the angiogenic switch through physical engrafting into the lumen of nascent vessels or paracrine release of pro-angiogenic factors. CBT is hampered by the paucity of EPCs harvested from peripheral blood and suffered from several pitfalls, including the differentiation outcome of transplanted cells and low percentage of engrafted cells. Therefore, CBT will benefit from a better understanding of the signal transduction pathway(s) which govern(s) EPC homing, proliferation and incorporation into injured tissues. At the same time, this information might outline alternative molecular targets to combat tumoral neovascularization. We have recently found that store-operated Ca(2+) entry, a Ca(2+)-permeable membrane pathway that is activated upon depletion of the inositol-1,4,5-trisphosphate-sensitive Ca(2+) pool, is recruited by vascular endothelial growth factor to support proliferation and tubulogenesis in human circulating endothelial colony forming cells (ECFCs). ECFCs are a subgroup of EPCs that circulate in the peripheral blood of adult individuals and are able to proliferate and differentiate into endothelial cells and form capillary networks in vitro and contribute to neovessel formation in vivo. The present review will discuss the relevance of SOCE to ECFC-based cell therapy and will address the pharmacological inhibition of store-dependent Ca(2+) channels as a promising target for anti-angiogenic treatments.

Endothelial progenitor cells (EPCs) home from the bone marrow to the site of tissue regeneration and sustain neovascularization after acute vascular injury and upon the angiogenic switch in solid tumors. Therefore, they represent a suitable tool for cell-based therapy (CBT) in regenerative medicine and provide a novel promising target in the fight against cancer. Intracellular Ca2+ signals regulate numerous endothelial functions, such as proliferation and tubulogenesis. The growth of endothelial colony forming cells (ECFCs), which are EPCs capable of acquiring a mature endothelial phenotype, is governed by store-dependent Ca2+ entry (SOCE). This study aimed at investigating the nature and the role of VEGF-elicited Ca2+ signals in ECFCs. VEGF induced asynchronous Ca2+ oscillations, whose latency, amplitude, and frequency were correlated to the growth factor dose. Removal of external Ca2+ (0Ca2+) and SOCE inhibition with N-(4-[3,5-bis(trifluoromethyl)-1H-pyrazol-1-yl]phenyl)-4-methyl-1,2,3-thiadiazole-5-carboxamide (BTP-2) reduced the duration of the oscillatory signal. Blockade of phospholipase C-γ with U73122, emptying the inositol-1,4,5-trisphosphate (InsP3)-sensitive Ca2+ pools with cyclopiazonic acid (CPA), and inhibition of InsP3 receptors with 2-APB prevented the Ca2+ response to VEGF. VEGF-induced ECFC proliferation and tubulogenesis were inhibited by the Ca2+-chelant, BAPTA, and BTP-2. NF-κB activation by VEGF was impaired by BAPTA, BTP-2, and its selective blocker, thymoquinone. Thymoquinone, in turn, suppressed VEGF-dependent ECFC proliferation and tubulogenesis. These data indicate that VEGF-induced Ca2+ oscillations require the interplay between InsP3-dependent Ca2+ release and SOCE, and promote ECFC growth and tubulogenesis by engaging NF-κB. This novel signaling pathway might be exploited to enhance the outcome of CBT and chemotherapy.

Background Extraovarian Primary Peritoneal Carcinoma (EOPPC) was first described by Swerdlow in 1959 [1]. Basically, EOPPC is a malignancy that spreads widely inside the peritoneal cavity involving mostly the omentum with minimal or no ovarian involvement. Most of the EOPPC cases reported have been of serous histology; histopathological, immunohistochemical, and clinical similarities have been observed between EOPPC and Epithelial Ovarian Cancer (EOC).

Endothelial progenitor cells (EPCs) may be recruited from the bone marrow to sites of tissue regeneration to sustain neovascularization and reendothelialization after acute vascular injury. This feature makes them particularly suitable for cell-based therapy. In mature endothelium, store-operated Ca(2+) entry (SOCE) is activated following emptying of inositol-1,4,5-trisphosphate-sensitive stores, and controls a wide number of functions, including proliferation, nitric oxide synthesis, and vascular permeability. The present work aimed at investigating SOCE expression in EPCs harvested from both peripheral blood (PB-EPCs) and umbilical cord blood (UCB-EPCs) by employing both Ca(2+) imaging and molecular biology techniques. SOCE was induced upon either pharmacological (ie, cyclopiazonic acid) or physiological (ie, ATP) depletion of the intracellular Ca(2+) pool. Further, store-dependent Ca(2+) entry was inhibited by the SOCE inhibitor, N-(4-[3,5-bis(trifluoromethyl)-1H-pyrazol-1-yl]phenyl)-4-methyl-1,2,3-thiadiazole-5-carboxamide (BTP-2). Real-time reverse transcription-polymerase chain reaction and western blot analyses showed that both PB-EPCs and UCB-EPCs express all the molecular candidates to mediate SOCE in differentiated cells, including TRPC1, TRPC4, Orai1, and Stim1. Moreover, pharmacological maneuvers demonstrated that, as well as in differentiated endothelial cells, the signal transduction pathway leading to depletion of the intracellular Ca(2+) pool impinged on the phospholipase C/inositol-1,4,5-trisphosphate pathway. Finally, blockage of SOCE with BTP-2 impaired PB-EPC proliferation. These findings provide the first evidence that EPCs express SOCE, which might thus be regarded as a novel target to enhance the regenerative outcome of cell-based therapy.

Cephalopods are endowed with the most sophisticated nervous system among invertebrates and exhibit a repertoire of complex behaviors, such as spatial and observational learning. Cephalopod eyes supply a wide range of information which are utilized for these learning behaviors. Although our understanding of vertebrate physiology greatly benefited from the sub-cellular analysis of cephalopod nervous system, as shown by the discovery of the ionic bases of action potentials and of the Ca2+ requirement for neurotransmitter release Surprisingly, the cellular basis by which the visual system drives the sophisticated repertoire of cephalopod behaviors are still poorly understood. In this review, we will describe the current knowledge about cephalopod phototransduction. Light excites cephalopod photoreceptors by either inducing Ca2+ release from intracellular stores or activating cation-permeable channels by an as yet unknown mechanism. A 92 kDa protein, which is homologous to the Drosophila transient receptor potential (TRP) gene, is the most likely mediator of light-induced currents in cephalopods. A number of models which explain the mechanism whereby cephalopod TRP channel is gated by light will be discussed.

Introduction Although laparoscopic Nissen fundoplication has been recognized as the standard of care for hiatal hernia (HH) repair, HH recurrence due to breakdown of the hiatoplasty have been reported as a common mechanism of failure after primary repair. Different surgical techniques for diaphragmatic pillars closure have been proposed, but the problem remains unsolved. The authors hypothesized that ultrastructural illness may be implicated in this recurrence. The aim of this study was to investigate the presence of changes at esophageal hiatal area in patients with and without HH. Materials and Methods One hundred and thirty-two laparoscopic samples from phrenoesophageal membrane and diaphragmatic crura were collected from 33 patients with gastroesophageal reflux disease and HH (HH group) and 60 samples from 15 patients without HH enrolled as the control group (NHH group). All specimens were processed and analyzed by transmission electron microscopy. Results Muscular and connective samples from the NHH group showed no ultrastructural alterations; similar results were found in phrenoesophageal ligament samples from the HH group. In contrast, 94% of the muscular samples obtained from the crura of the HH group have documented four main types of alterations. In 75% of HH patients, the pillar lesions were severe. Conclusion Patients with hiatal hernia have ultrastructural abnormalities at the muscular tissue of the crura that are not present in patients with a normal gastroesophageal junction. There is no difference in the microscopic damage at the connective tissue of the phrenoesophageal membrane surrounding the esophagus of the two groups of patients. The outcome of antireflux surgery could depend not only on the adopted surgical technique but also on the underlying status of the diaphragmatic crura.

Incisional hernia (IH) is an abdominal hernia that occurs after previous surgery [1]. It is one of the most frequent surgical problems, as the incidence following laparotomy is 10–20% [2, 3, 4, 5]. Surgical treatment of incisional hernia involves either direct suture in patients with primary defects >3 cm in diameter, or a rather complex operation in patients with large defects that involve loss of wall substance and impairment of ventilation and cardiac circulation.

Synaptic transmission was examined in the plexiform zone of Octopus vulgaris optic lobes using field-potential recording from optic lobe slices. Stimulation of the optic nerve produced pre- and postsynaptic field potentials. Transmission was abolished in calcium-free seawater, L- glutamate or the AMPA/Kainate receptor blocker CNQX (EC(50), 40 microm), leaving an intact presynaptic field potential. ACh markedly reduced or blocked and d-tubocurarine augmented both pre- and postsynaptic field potentials, while alpha-bungarotoxin and atropine were without effect. Paired-pulse stimulation showed short-term depression of pre- and postsynaptic components with a half-time of recovery of approximately 500 ms. The depression was partially relieved in the presence of d-tubocurarine (half-time of recovery, 350 ms). No long-term changes in synaptic strength were induced by repetitive stimulation. A polyclonal antibody raised against a squid glutamate receptor produced positive staining in the third radial layer of the plexiform zone. No positive staining was observed in the other layers. Taking into account previous morphological data and our results, we propose that the excitatory terminations of the photoreceptors are in the innermost layer of the plexiform zone where the transmitter is likely to be glutamate and postsynaptic receptors are AMPA/kainate-like. Thus, the function of the terminal bags is to provide a location for a presynaptic cholinergic inhibitory shunt. The results imply that this arrangement provides a temporal filter for visual processing and enhances the perception of moving vs. stationary objects.

The mechanism whereby extracellular Ca(2+) exerts the endothelium-dependent control of vascular tone is still unclear. In this study, we assessed whether cardiac microvascular endothelial cells (CMEC) express a functional extracellular Ca(2+)-sensing receptor (CaSR) using a variety of techniques. CaSR mRNA was detected using RT-PCR, and CaSR protein was identified by immunocytochemical analysis. In order to assess the functionality of the receptor, CMEC were loaded with the Ca(2+)-sensitive fluorochrome, Fura-2/AM. A number of CaSR agonists, such as spermine, Gd(3+), La(3+) and neomycin, elicited a heterogeneous intracellular Ca(2+) signal, which was abolished by disruption of inositol 1,4,5-trisphosphate (InsP(3)) signaling and by depletion of intracellular stores with cyclopiazonic acid. The inhibition of the Na(+)/Ca(2+) exchanger upon substitution of extracellular Na(+) unmasked the Ca(2+) signal triggered by an increase in extracellular Ca(2+) levels. Finally, aromatic amino acids, which function as allosteric activators of CaSR, potentiated the Ca(2+) response to the CaSR agonist La(3+). These data provide evidence that CMEC express CaSR, which is able to respond to physiological agonists by mobilizing Ca(2+) from intracellular InsP(3)-sensitive stores.

Depolymerization of the actin cytoskeleton may liberate Ca2+ from InsP3-sensitive stores in some cell types, including starfish oocytes, while inhibiting Ca2+ influx in others. However, no information is available on the modulation of membrane potential (V(m)) by actin. The present study was aimed to ascertain whether the widely employed actin depolymerizing drug, latrunculin A (Lat A), affects V(m) in mature oocytes of the starfish Astropecten aranciacus. Lat A induced a membrane depolarization which was mimicked by cytochalasin D, another popular actin disruptor, and prevented by jasplakinolide, a stabilizer of the actin network. Lat A-elicited depolarization consisted in a positive shift in V(m) which reached the threshold of activation of voltage-gated Ca2+ channels (VGCC), thus triggering an action potential. Lat A-promoted depolarization lacked the action potential in Ca2+-free sea water, while it was abolished upon removal of external Na+. Moreover, membrane depolarization was prevented by pre-injection of BAPTA and heparin, but not ryanodine. These data indicate that Lat A induces a membrane depolarization by releasing Ca2+ from InsP3Rs. The Ca2+ signal in turn activates a Ca2+-dependent Na+ entry, which causes the positive shift in V(m) and stimulates the VGCC.

Summary The inner wall of excised rat aorta was scraped by a microelectrode and Ca 2+ signals were investigated by fluorescence microscopy in endothelial cells (ECs) directly coupled with injured cells. The injury caused an immediate increase in the intracellular Ca 2+ concentration ([Ca 2+ ] i ), followed by a long-lasting decay phase due to Ca 2+ influx from extracellular space. The immediate response was mainly due to activation of purinergic receptors, as shown by the effect of P 2X and P 2Y receptors agonists and antagonists, such as suramin, α,β-MeATP, MRS-2179 and 2-MeSAMP. Inhibition of store-operated Ca 2+ influx did not affect either the peak response or the decay phase. Furthermore, the latter was: (i) insensitive to phospholipase C inhibition, (ii) sensitive to the gap junction blockers, palmitoleic acid, heptanol, octanol and oleamide, and (iii) sensitive to La 3+ and Ni 2+ , but not to Gd 3+ . Finally, ethidium bromide or Lucifer Yellow did not enter ECs facing the scraped area. These results suggest that endothelium scraping: (i) causes a short-lasting stimulation of healthy ECs by extracellular nucleotides released from damaged cells and (ii) uncouples the hemichannels of the ECs facing the injury site; these hemichannels do not fully close and allow a long-lasting Ca 2+ entry.

The recently discovered second messenger nicotinic acid adenine dinucleotide phosphate (NAADP) is central to the onset of intracellular Ca2+ signals induced by several stimuli, including fertilization. The nature of the Ca2+ pool mobilized by NAADP is still controversial. Depending on the cell type, NAADP may target either an acidic compartment with lysosomal properties or ryanodine receptors (RyRs) on endoplasmic reticulum. In addition, NAADP elicits a robust Ca2+ influx into starfish oocytes by activating a Ca2+-mediated current across the plasma membrane. In the present study, we employed the single-electrode intracellular recording technique to assess the involvement of either acidic organelles or RyRs in NAADP-elicited Ca2+ entry. We found that neither drugs which interfere with acidic compartments nor inhibitors of RyRs affected NAADP-induced depolarization. These data further support the hypothesis that a yet unidentified plasma membrane Ca2+ channel is the target of NAADP in starfish oocytes.

The expression and function of nicotinic ACh receptors (nAChRs) in rat coronary microvascular endothelial cells (CMECs) were examined using RT-PCR and whole cell patch-clamp recording methods. RT-PCR revealed expression of mRNA encoding for the subunits α2, α3, α4, α5, α7, β2, and β4 but not β3. Focal application of ACh evoked an inward current in isolated CMECs voltage clamped at negative membrane potentials. The current-voltage relationship of the ACh-induced current exhibited marked inward rectification and a reversal potential ( Erev) close to 0 mV. The cholinergic agonists nicotine, epibatidine, and cytisine activated membrane currents similar to those evoked by ACh. The nicotine-induced current was abolished by the neuronal nAChR antagonist mecamylamine. The direction and magnitude of the shift in Erev of nicotine-induced current as a function of extracellular Na+ concentration indicate that the nAChR channel is cation selective and follows that predicted by the Goldman-Hodgkin-Katz equation assuming K+/Na+ permeability ratio of 1.11. In fura-2-loaded CMECs, application of ACh, but not of nicotine, elicited a transient increase in intracellular free Ca2+ concentration. Taken together, these results demonstrate that neuronal nAChR activation by cholinergic agonists evokes an inward current in CMECs carried primarily by Na+, which may contribute to the plasma nicotine-induced changes in microvascular permeability and reactivity induced by elevations in plasma nicotine.

Cyclic ADP-ribose (cADPr) is a second messenger that regulates intracellular free [Ca2+] ([Ca2+](i)) in a variety of cell types, including immature oocytes from the starfish Astropecten auranciacus. In this study, we employed confocal laser scanning microscopy and voltage clamp techniques to investigate the source of the cADPr-elicited Ca2+ wave originating from the cortical Ca2+ patches we have described previously. The Ca2+ swing was accompanied by a membrane current with a reversal potential of approximately +20 mV. Decreasing external Na+ almost abolished the current without affecting the Ca2+ response. Removal of extracellular Ca2+ altered neither the Ca2+ transient nor the ionic current, nor did the holding potential exert any effect on the Ca2+ wave. Both the Ca2+ response and the membrane current were abolished when BAPTA, ruthenium red or 8-NH(2)-cADPr were preinjected into the oocytes, while perfusion with ADPr did not elicit any [Ca2+](i) increase or ionic current. However, elevating [Ca2+](i) by uncaging Ca2+ from nitrophenyl- (NP-EGTA) or by photoliberating inositol 1,4,5-trisphosphate (InsP(3)) induced an ionic current with biophysical properties similar to that elicited by cADPr. These results suggest that cADPr activates a Ca2+ wave by releasing Ca2+ from intracellular ryanodine receptors and that the rise in [Ca2+](i) triggers a non-selective monovalent cation current that does not seem to contribute to the global Ca2+ elevation.

An increase in intracellular Ca 2 + concentration ([Ca 2 + ] i ) may play a role in the proliferative effect of several growth factors. In this study, the changes in [Ca 2 + ] i elicited by epidermal growth factor (EGF) in rat cardiac microvascular endothelial cells (CMEC) have been investigated by using fura-2 conventional and confocal microscopy. A large heterogeneity in the latency and in the pattern of the Ca 2 + response was found at each dose of EGF (2.5-100 ng/ml), whereas some cells displayed a non-oscillatory behavior and others exhibited a variable number of Ca 2 + oscillations. On average, the fraction of responsive cells, the total number of oscillations and the duration of the Ca 2 + signal were higher at around 10 ng/ml EGF, while there was no dose-dependence in the lag time and in the amplitude of the [Ca 2 + ] i increase. EGF-induced Ca 2 + spikes were abolished by the tyrosine kinase inhibitor genistein, but not by its inactive analogue daidzein, and by the phospholipase C blocker NCDC. Only 1-2 transients could be elicited in Ca 2 + -free solution, while re-addition of extracellular Ca 2 + recovered the spiking activity. The oscillatory signal was prevented by the SERCA inhibitor thapsigargin and abolished by the calcium entry blockers Ni 2 + and La 3 + . Moreover, EGF-induced Ca 2 + transients were abolished by the InsP 3 receptor blocker caffeine, while ryanodine was without effect. Confocal imaging microscopy showed that the Ca 2 + response to EGF was localized both in the cytoplasm and in the nucleus. We suggest that EGF-induced [Ca 2 + ] i increase may play a role in the proliferative action of EGF on endothelial cells.

In cardiac cells that lack macroscopic transient outward K+ currents ( I to), the removal of extracellular Ca2+ can unmask “ I to-like” currents. With the use of pig ventricular myocytes and the whole cell patch-clamp technique, we examined the possibility that cation efflux via L-type Ca2+channels underlies these currents. Removal of extracellular Ca2+ and extracellular Mg2+ induced time-independent currents at all potentials and time-dependent currents at potentials greater than −50 mV. Either K+ or Cs+ could carry the time-dependent currents, with reversal potential of +8 mV with internal K+ and +34 mV with Cs+. Activation and inactivation were voltage dependent [Boltzmann distributions with potential of half-maximal value ( V 1/2) = −24 mV and slope = −9 mV for activation; V 1/2 = −58 mV and slope = 13 mV for inactivation]. The time-dependent currents were resistant to 4-aminopyridine and to DIDS but blocked by nifedipine at high concentrations (IC50 = 2 μM) as well as by verapamil and diltiazem. They could be increased by BAY K-8644 or by isoproterenol. We conclude that the I to-like currents are due to monovalent cation flow through L-type Ca2+ channels, which in pig myocytes show low sensitivity to nifedipine.

Intracellular Ca 2+ signals elicited by nucleotide agonists were investigated in primary cultures of rat cardiac microvascular endothelial cells using the fura-2 technique. UTP increased the intracellular [Ca 2+ ] in 94% of the cells, whereas 2MeSATP was active in 32%. The rank order of potency was ATP = UTP > 2MeSATP and the maximal response to 2MeSATP was lower compared to UTP and ATP. ATP and UTP showed strong homologous and heterologous desensitization. ATP fully inhibited the 2MeSATP response, while UTP abolished 2MeSATP-elicited transients in 25% of cells. 2MeSATP did not desensitize the UTP or ATP response. Adenosine 2′,5′-diphosphate inhibited the response to 2MeSATP, while it did not modify the response to ATP and UTP. 2MeSATP was more sensitive to suramin than UTP and ATP. These results indicate that P 2Y1 and P 2Y2 receptors may be coexpressed in CMEC. Nucleotide-induced Ca 2+ signals lacked a sustained plateau and were almost independent from extracellular Ca 2+ . ATP and UTP elicited Ca 2+ transients longer than 2MeSATP-evoked transients. The kinetics of Ca 2+ responses was not affected by bath solution stirring or ectonucleotidase inhibition. Furthermore, the nonhydrolyzable ATP analogue AMP-PNP induced Ca 2+ signals similar to those elicited by ATP and UTP. These results suggest that the distinct kinetics of nucleotide-evoked Ca 2+ responses do not depend on the activity of ectonucleotidases or ATP autocrine stimulation. The possibility that Ca 2+ signals with different time courses may modulate different cellular responses is discussed.

The patch-clamp technique was used to investigate the electrical response of cardiac microvascular endothelial cells to fluid stream applied through a microtube. The fluid stream induced a membrane current whose amplitude was dependent on flow rate. The I-V relationship of the flow-activated current showed a strong inward rectification and a reversal potential close to +30 mV, which gave a P(Na)/P(k)=3.47. The inward and outward components of the current nearly disappeared when extracellular Na(+)and internal K(+), respectively, were replaced by NMDG(+). Finally, the flow-activated current was fully blocked by 50 microM Gd(3+)and La(3+). These results show that cardiac microvascular endothelial cells respond to mechanical stimulation associated with flow by increasing their permeability to Na(+)and K(+). This mechanism is suggested to contribute to ionic homeostasis at high heart rates and during post-ischaemic reperfusion.

Background The study was conducted on a population of 74 patients undergoing surgery for rectal adenocarcinoma with 5-year follow-up. The aim is to evaluate the survival and Quality of Life (QoL) in two groups of patients: Y group with age

Introduction Among the surgical procedures for treatment of morbid obesity, laparoscopic sleeve gastrectomy has known widespread diffusion in the last years, although it is not free from significant morbidity rates. Aim of this work is to evaluate the incidence of Helicobacter pylori (HP) infection on the postoperative outcome of patients undergoing laparoscopic sleeve gastrectomy. Methods Between January 2008 and December 2013, 184 patients (65 males, 119 females), mean age 35.8 ± 5.7 years, affected with morbid obesity, mean BMI 46.6 ± 6.7, underwent laparoscopic sleeve gastrectomy. All the specimens at the end of the operation were analysed by the same pathologist. Histological grading was based on the Sidney classification. Results Seventy-two of the patients (39.1%) were HP positive, while 112 (60.9%) were negative. No significant differences were observed between the HP+ and HP− group in terms of age, sex, weight, BMI, incidence of comorbidities and duration of follow-up. All the operations were completed via laparoscopic approach. No mortality was observed. Postoperative complications occurred in 5 patients (2.7%): three leaks (1.6%), all in the HP- group and two bleedings (1.1%), one in the HP+ and one in the HP− group. In two cases a reintervention was necessary. No significant differences were observed in the morbidity rates between the two groups. Overall mean excess weight loss at 6 months, 12 months and 24 months was respectively 47.4 ± 11.3%, 61.1 ± 12.4% and 68.4 ± 13.5%, with no significant differences between the HP+ and HP− groups. Conclusions HP infection seems not to influence postoperative outcome of patients operated of laparoscopic sleeve gastrectomy.

Endothelial progenitor cells (EPCs) may be recruited from bone marrow to sustain the metastatic switch in a number of solid cancers, including breast cancer (BC) and renal cellular carcinoma (RCC). Preventing EPC mobilization causes tumor shrinkage. Novel anti-angiogenic treatments have been introduced in therapy to inhibit VEGFR-2 signaling; unfortunately, these drugs blocked tumor angiogenesis in pre-clinical murine models, but resulted far less effective in human patients. Understanding the molecular mechanisms driving EPC proliferation and tubulogenesis in cancer patients could outline novel targets for alternative anti-angiogenic treatments. Store-operated Ca2 + entry (SOCE) regulates the growth of human EPCs, and it is mediated by the interaction between the endoplasmic reticulum Ca2 +-sensor, Stim1, and the plasmalemmal Ca2 + channels, Orai1 and TRPC1. EPCs do not belong to the neoplastic clone: thus, unlike tumor endothelium and neoplastic cells, they should not remodel their Ca2 + toolkit in response to tumor microenvironment. However, our recent work demonstrated that EPCs isolated from naive RCC patients (RCC-EPCs) undergo a dramatic remodeling of their Ca2 + toolkit by displaying a remarkable drop in the endoplasmic reticulum Ca2 + content, by down-regulating the expression of inositol-1,4,5-receptors (InsP3Rs), and by up-regulating Stim1, Orai1 and TRPC1. Moreover, EPCs are dramatically less sensitive to VEGF stimulation both in terms of Ca2 + signaling and of gene expression when isolated from tumor patients. Conversely, the pharmacological abolition of SOCE suppresses proliferation in these cells. These results question the suitability of VEGFR-2 as a therapeutically relevant target for anti-angiogenic treatments and hint at Orai1 and TRPC1 as more promising alternatives. This article is part of a Special Issue entitled: 13th European Symposium on Calcium.

This paper investigates the phenomenon of cover spalling in reinforced concrete induced by bond or by the action of an inner pressure. This research is based on an experimental programme comprising a series of bond tests and a series of tests with inner-pressure on cylindrical openings. The inner-pressure test series (aimed at representing the conditions occurring for instance due to corrosion of reinforcement) was performed with hydraulic inflator devices embedded within concrete openings near to the free concrete surface. By means of detailed surface measurements performed with Digital Image Correlation, the mechanisms triggering spalling failures and the associated resistance are discussed and analysed thoroughly. This series investigates in addition a number of phenomena relevant to spalling failures, such as the influence of the casting position, group and size effects. The observed response, analysed by means of a mechanical analogy, is later used to investigate a series of structural tests performed on pull-out specimens. This analysis highlights the analogies and differences between the two types of tests (subjected to an imposed pressure or to bond stresses). On that basis, a comprehensive approach for treatment of bond-related cases failing by cover spalling is proposed, showing consistent agreement to the experimental evidence.

It is well known that control specimens used to assess the concrete strength of new structures have different casting and curing conditions than those of actual structures. Notably, after pouring of the concrete and before its hardening, a number of phenomena such as concrete bleeding and plastic settlement occur, influencing the in-situ strength with respect to that of small and homogeneous control specimens (cubes or cylinders). In addition, the development of these phenomena and their structural implications are influenced by the presence of reinforcing bars, disturbing the settlement and bleeding of fresh concrete. In this paper, these aspects, with particular emphasis on the effective structural strength, are investigated by means of a testing program performed with refined measurement techniques such as tomography and Digital Image Correlation. On that basis, consistent design rules are derived to correct the strength of control specimens in order to calculate the resistance of a structural concrete member.

Hematopoietic progenitor and stem cells (HPSCs) have been employed in cell-based therapy (CBT) to promote neovascularisation and regeneration of ischemic organs, such as heart and limbs. Furthermore, endothelial progenitor cells (EPCs) may favour tumour growth and adverse vascular targeting treatment by incorporating into neovessels. CBT is hampered by the paucity of HPSCs harvested from peripheral blood and suffers from several pitfalls, including the differentiation outcome of transplanted cells and low percentage of engrafted cells. Therefore, CBT will benefit of a better understanding of the signal transduction pathway(s) which drive(s) HPSC homing, proliferation and incorporation into injured tissues. At the same time, this information might outline alternative molecular targets to combat tumoral neovascularisation. The elevation in intracellular Ca2+ concentration is the key signal in the regulation of cellular motility, replication, and differentiation. Intracellular Ca2+ waves regulate cytoskeleton re-organisation and disassembly at focal adhesions, thus stimulating migration and substrate adhesion, and induce DNA transcription by recruiting Ca2+-sensitive transcription factors. However, the Ca2+ signalling toolkit which underlies Ca2+ release from intracellular stores and Ca2+ entry across the plasmalemma in HPSCs is still unclear. Our recent work has shown that the so-called store-operated Ca2+ entry stimulates EPC growth. Unravelling the mechanisms guiding HPSC behaviour might supply the biological bases required to improve CBT. For instance, genetic manipulation of the Ca2+ signalling machinery (such as transfer of genes encoding for the Ca2+ channels involved in EPC proliferation) could provide a novel approach to increase the extent of limb neovascularisation and regeneration of damaged hearts.

Starfish oocytes that are extracted from the ovaries are arrested at the prophase of the first meiotic division. At this stage of maturation, they are characterized by a large nucleus called the germinal vesicle. Meiosis resumption (maturation) can be induced in vitro by adding the hormone 1-methyladenine (1-MA) to the seawater in which the oocytes are suspended. Earlier work in our laboratory had detected Ca(2+) increases in both the cytoplasm and the nucleus of the oocytes approx. 2 min after the 1-MA challenge. The nuclear Ca(2+) increase was found to be essential for the continuation of the meiotic cycle, since the injection of bis-(o-aminophenoxy)ethane- N,N,N',N' -tetra-acetic acid (BAPTA) into the nuclear compartment completely blocked the re-initiation of the cell cycle. We have recently confirmed, using confocal microscopy, that the cytoplasmic and nuclear Ca(2+) pools are regulated independently and that the nuclear envelope in starfish oocytes is not freely permeated by the Ca(2+) wave that sweeps across the nuclear region. Studies by others have shown that the sensitivity of the Ins(1,4,5) P (3) (IP(3)) receptors (IP(3)Rs) to IP(3) increases during oocyte maturation, so that they release progressively more calcium in response to the injection of IP(3), as maturation proceeds. We have now shown that the increased sensitivity of the IP(3)Rs may depend on the activation of the cyclin-dependent kinase, MPF (M-phase-promoting factor) that occurs in the nucleus. MPF does not directly phosphorylate IP(3)Rs but phosphorylates instead the actin-binding protein actin depolymerization factor (ADF)/cofilin.

The endothelium is a cellular monolayer that lines the inner surface of blood vessels and plays a central role in the maintenance of cardiovascular homeostasis by controlling platelet aggregation, vascular tone, blood fluidity and fibrinolysis, adhesion and transmigration of inflammatory cells, and angiogenesis. Endothelial dysfunctions are associated with various cardiovascular diseases, including atherosclerosis, hypertension, myocardial infarction, and cardiovascular complications of diabetes. Numerous studies have established the anti-inflammatory, anti-apoptotic, and anti-oxidant effects of hydrogen sulfide (H2S), the latest member to join the gasotransmitter family along with nitric oxide and carbon monoxide, on vascular endothelium. In addition, H2S may prime endothelial cells (ECs) toward angiogenesis and contribute to wound healing, besides to its well-known ability to relax vascular smooth muscle cells (VSMCs), and thereby reducing blood pressure. Finally, H2S may inhibit VSMC proliferation and platelet aggregation. Consistently, a deficit in H2S homeostasis is involved in the pathogenesis of atherosclerosis and of hyperglycaemic endothelial injury. Therefore, the application of H2S-releasing drugs or using gene therapy to increase endogenous H2S level may help restore endothelial function and antagonize the progression of cardiovascular diseases. The present article reviews recent studies on the role of H2S in endothelial homeostasis, under both physiological and pathological conditions, and its putative therapeutic applications.

Cardiovascular disease (CVD) comprises a range of major clinical cardiac and circulatory diseases, which produce immense health and economic burdens worldwide. Currently, vascular regenerative surgery represents the most employed therapeutic option to treat ischemic disorders, even though not all the patients are amenable to surgical revascularization. Therefore, more efficient therapeutic approaches are urgently required to promote neovascularization. Therapeutic angiogenesis represents an emerging strategy that aims at reconstructing the damaged vascular network by stimulating local angiogenesis and/or promoting de novo blood vessel formation according to a process known as vasculogenesis. In turn, circulating endothelial colony-forming cells (ECFCs) represent truly endothelial precursors, which display high clonogenic potential and have the documented ability to originate de novo blood vessels in vivo. Therefore, ECFCs are regarded as the most promising cellular candidate to promote therapeutic angiogenesis in patients suffering from CVD. The current briefly summarizes the available information about the origin and characterization of ECFCs and then widely illustrates the preclinical studies that assessed their regenerative efficacy in a variety of ischemic disorders, including acute myocardial infarction, peripheral artery disease, ischemic brain disease, and retinopathy. Then, we describe the most common pharmacological, genetic, and epigenetic strategies employed to enhance the vasoreparative potential of autologous ECFCs by manipulating crucial pro-angiogenic signaling pathways, e.g., extracellular-signal regulated kinase/Akt, phosphoinositide 3-kinase, and Ca2+signaling. We conclude by discussing the possibility of targeting circulating ECFCs to rescue their dysfunctional phenotype and promote neovascularization in the presence of CVD.

In non-excitable cells, many agonists increase the intracellular Ca(2+) concentration ([Ca(2+)](i)) by inducing an inositol 1,4,5-trisphosphate (IP(3))-mediated Ca(2+) release from the intracellular stores. Ca(2+) influx from the extracellular medium may then sustain the Ca(2+) signal. [Ca(2+)](i) recovers its resting level as a consequence of Ca(2+)-removing mechanisms, i.e. plasma-membrane Ca(2+)-ATPase (PMCA) pump, Na(+)/Ca(2+) exchanger (NCX) and sarco-endoplasmic reticulum Ca(2+)-ATPase (SERCA) pump. In a study performed in pancreatic acinar cells, evidence has been provided suggesting that, during the decay phase of the agonist-evoked Ca(2+) transients, the Ca(2+) concentration within the intracellular stores remains essentially constant [Mogami, Tepikin and Petersen (1998) EMBO J. 17, 435-442]. It was therefore hypothesized that, in such a situation, intracellular Ca(2+) is not only picked up by the SERCA pump, but is also newly released through IP(3)-sensitive Ca(2+) channels, with the balance between these two processes being approximately null. The main aim of the present work was to test this hypothesis by a different experimental approach. Using cardiac microvascular endothelial cells, we found that inhibition of the SERCA pump has no effect on the time course of agonist-evoked Ca(2+) transients. This result was not due to a low capacity of the SERCA pump since, after agonist removal, this pump proved to be very powerful in clearing the excess of intracellular Ca(2+). We showed further that: (i) in order to avoid a rapid removal of Ca(2+) by the SERCA pump, continuous IP(3) production appears to be required throughout all of the decay phase of the Ca(2+) transient; and (ii) Ca(2+) picked up by the SERCA pump can be fully and immediately released by agonist application. All these results support the model of Mogami, Tepikin and Petersen [(1998) EMBO J. 17, 435-442]. Since the SERCA pump did not appear to be involved in shaping the decay phase of the agonist-evoked Ca(2+) transient, we inhibited the PMCA pump with carboxyeosin, and NCX with benzamil and by removing extracellular Na(+). The results indicate that, during the decay phase of the agonist-evoked Ca(2+) transient, the intracellular Ca(2+) is removed by both the PMCA pump and NCX. Finally, we provide evidence indicating that mitochondria have no role in clearing intracellular Ca(2+) during agonist-evoked Ca(2+) transients.

BACKGROUND In this prospective non-randomized observational cohort study we evaluated: the feasibility and effectiveness of primary umbilical hernia repair with open tension-free and sutureless technique using a porcine small intestinal submucosa (Surgisis) prosthesis, the quality of the treatment in terms of reduction of postoperative discomfort and the complications at early and long-term follow-up. METHODS Thirty-six consecutive patients, mean age 45.25 +/- 12.19 years, affected by primary umbilical uncomplicated hernia with a defect size < or = 3 cm, were treated in a day-surgery setting. A tailored flat Surgisis graft was used to ensure an overlap of at least 2 cm; in all patients the mesh was fixed by fibrin glue. Collected data included: visual analogic scale (VAS) pain scores at 24 hours, 72 hours, and 7, 15, and 30 days and number of analgesic medications after operation, complications rate, the quality of life measured by Short Form 36 health survey questionnaire (SF-36) before the operation and at long term follow-up. RESULTS The mean follow-up time was 5.6 +/- 1.4 years. Postoperative pain was low: the mean visual analogic scale (VAS) scores were 2.8 at 24 h, 1.8 at 72 h, and 0.9, 0.3, and 0.04 at 7, 15, and 30 days, respectively. 77.8% of the patients (28/36) did not use any analgesic drugs. Seroma was reported in 13.8% of the patients (5/36); there were no hematomas, infection, chronic pain and no major complications or mortality (< or = 30 days). Recurrence rate was 2.8% (1/36). Patient satisfaction showed a significant improvement in all SF-36 domain scores (P < 0.001). CONCLUSIONS The biologic mesh seems to be a safe and reliable device for repairing primary umbilical hernia with high patient comfort, even if not yet an alternative to synthetic mesh.

Background: In this prospective non randomized observational cohort study we have evaluated the influence of age on outcome of laparoscopic total fundoplication for GERD. Methods: Six hundred and twenty consecutive patients underwent total laparoscopic fundoplication for GERD. Five hundred and twenty-four patients were younger than 65 years (YG), and 96 patients were 65 years or older (EG). The following parameters were considered in the preoperative and postoperative evaluation: presence, duration, and severity of GERD symptoms, presence of a hiatal hernia, manometric and 24 hour pH-monitoring data, duration of operation, incidence of complications and length of hospital stay. Results: Elderly patients more often had atypical symptoms of GERD and at manometric evaluation had a higher rate of impaired esophageal peristalsis in comparison with younger patients. The duration of the operation was similar between the two groups. The incidence of intraoperative and postoperative complications was low and the difference was not statistically significant between the two groups. An excellent outcome was observed in 93.0% of young patients and in 88.9% of elderly patients (p = NS). Conclusions: Laparoscopic antireflux surgery is a safe and effective treatment for GERD even in elderly patients, warranting low morbidity and mortality rates and a significant improvement of symptoms comparable to younger patients.

Background Postoperative complications such as failure or disruption of the crura repair and intrathoracic migration of the wrap are the most common anatomic reasons for the failure of Laparoscopic Nissen Fundoplication. The authors hypothesized that ultrastructural illness may be implicated in this recurrence. The aim of this study was to investigate the presence of changes at esophageal hiatal area in patients with and without HH and to shed some light on the use of mesh in this surgery.

Retroperitoneal schwannomas are rare tumours originating from Schwann cells of peripheral nerve sheaths. Their clinical presentation is often delayed as they grow to a large size in a non-restrictive space, such as the retroperitoneum, before any clear symptomatology is manifested. Furthermore, the symptoms may mimic different diseases and be aspecific. The preoperative diagnosis is often unclear as no pathognomonic radiological features are known. Fine needle biopsy is not diagnostic due to tumour cell pleomorphism. We report the case of a 51-year-old woman presenting with unusual symptoms such as severe constipation, bowel distension, excess flatus, postprandial fullness and abdominal pain due to left colon compression by a large retroperitoneal tumour. A complete resection of the mass combined with sparing of the surrounding tissues was carried out through a midline laparotomy. Microscopic evaluation and immunohistochemistry documented a benign retroperitoneal schwannoma. Postoperatively, complete resolution of abdominal symptoms and no major complications were observed. At 28 months' follow-up no local recurrence was found. To the best of our knowledge, this is a rare case of retroperitoneal schwannoma with definite abdominal symptoms and with sudden onset of severe constipation.