Urethral reconstruction is one of the great surgical challenges for urologists. A cell-based tissue-engineered urethra may be an alternative for patients who have complicated long strictures and need urethral reconstruction. Here, we demonstrated the feasibility of using autologous urine-derived stem cells (USCs) seeded on small intestinal submucosa (SIS) to repair a urethral defect in a rabbit model.n = 12 for each group). A series of tests, including a retrograde urethrogram, histological analysis, and immunofluorescence, was undertaken 2, 3, 4, and 12 weeks after the operation to evaluate the effect of the autologous USCs on urethral reconstruction.Autologous USCs were obtained and characterized, and their capacity to differentiate into urothelial cells (UCs) and smooth muscle cells (SMCs) was tested. Then, USCs were labeled with PKH67, seeded on SIS, and transplanted to repair a urethral defect. The urethral defect model was surgically established in New Zealand white male rabbits. A ventral urethral gap was created, and the urethral mucosa was completely removed, with a mean rabbit penile urethra length of 2 cm. The urethral mucosal defect was repaired with a SIS scaffold (control group: SIS with no USCs; experimental group: autologous USC-seeded SIS; Autologous USCs could be easily collected and induced to differentiate into UCs and SMCs. In addition, the urethral caliber, speed of urothelial regeneration, content of smooth muscle, and vessel density were significantly improved in the group with autologous USC-seeded SIS. Moreover, inflammatory cell infiltration and fibrosis were found in the control group with only SIS, but not in the experimental autologous USC-seeded SIS group. Furthermore, immunofluorescence staining demonstrated that the transplanted USCs differentiated into UCs and SMCs in vivo.Autologous USCs can be used as an alternative cell source for cell-based tissue engineering for urethral reconstruction. Reconstruction of the adult and pediatric male penile urethra has been one of the great surgical challenges for urologists, especially in cases of recurrent hypospadias and long urethral defects after trauma. More than 200 different reconstructive methods for urethral stricture disease or congenital absence have been described, with a variety of donor tissues, such as free skin grafts , bladderThe use of tissue-engineered grafts to repair urethral defects is considered to be an approach with therapeutic potential. Natural collagen materials, such as bladder submucosa (BSM) , 9, smalAnother critical element required for successful urethral tissue engineering is the cell source. A subpopulation of stem cells can be easily isolated from human voided urine –17, i.e.Therefore, we cultured autologous rabbit USCs before surgery and seeded them onto a 5% PAA-treated SIS scaffold to reconstruct the urethra in rabbits. This cell-based tissue-engineered urethra may be useful to patients with complicated long strictures who need urethral reconstruction.Fresh porcine small intestines were obtained and chemically modified according to a protocol published previously by Liu et al. . The mucFresh SIS and 5% PAA-treated SIS were fixed in 2.5% glutaraldehyde. The SIS samples were frozen at –80 °C for 24 h, sputter-coated with gold, and mounted for analysis. Electron micrographs of mucosa slides were obtained with an acceleration voltage of 25.0 kV at a magnification of 400× using a Hitachi S-3000 N Scanning Electron Microscope .For measurement of residual DNA, fresh SIS and 5% PAA-treated SIS samples were dabbed dry with tissue paper and weighed. Samples were minced, and any remaining nucleic acids were extracted using a commercially available kit . The DNA concentration in each sample was calculated using a UV spectrophotometer  to measure the absorbance at 260 nm. The DNA yield was normalized to the initial wet weight of the samples.Urine and bladder irrigation solution samples  were collected from every rabbit that was to undergo surgery. Each sample was centrifuged at 500 g for 5 min to collect the cells, and the supernatant was carefully discarded. The pelleted cells were gently resuspended in medium (a 1:1 mixture of KSFM and embryonic fibroblast medium (EFM)) and plated in 24-well plates for single-cell cultures. The medium was changed every 2 days after the initial 72 h, and cells were passaged at 70–80% confluency .2 in medium composed of equal amounts of KSFM and EFM containing 2% serum and 30 ng/ml epidermal growth factor  and cultured for 14 days 20110016). Twenty-four New Zealand white male rabbits (2.0–2.5 kg) were randomly divided into two groups, each containing 12 animals. Autologous USCs had been obtained from each of these rabbits and cultured. The experimental group received urethroplasty with an autologous USC-seeded 5% PAA-treated SIS scaffold, and the control group received urethroplasty with a 5% PAA-treated SIS scaffold alone. Animals were anesthetized with pentobarbital (30 mg/kg) administered intravenously and lidocaine (6 mg/kg) administered subcutaneously to decrease any pain during and after the surgery. A ventral urethral gap was created a mean of 2 cm from the external urethral orifice, and the urethral mucosa was exposed and completely removed, with a mean penile urethra length of 2 cm. The urethral mucosal defect was repaired with a SIS scaffold  staining, 4′,6-diamidino-2-phenylindole (DAPI) staining, Masson’s trichrome staining, immunohistochemistry (IHC), and IF). IHC was performed using monoclonal antibodies against the urothelium , smooth muscle , and vascular endothelium . Briefly, consecutive 4.0-μm sections were prepared using a cryostat and incubated overnight at 4 °C with the following primary antibodies: anti-AE1/AE3 diluted 1:75, anti-myosin diluted 1:100, and anti-CD31 diluted 1:150. Then, sections were washed with phosphate-buffered saline (PBS) and incubated with the appropriate secondary antibody diluted 1:200 for 60 min at room temperature. Finally, the slides were rinsed twice with PBS and counterstained with hematoxylin or DAPI. IF was performed using monoclonal antibodies against the urothelium  and smooth muscle . Briefly, frozen sections (6.0 μm) or cells were fixed in 4% paraformaldehyde and blocked with 5% bovine serum albumin. Subsequently, the sections were treated overnight at 4 °C with the following primary antibodies: anti-AE1/AE3 diluted 1:75 or anti-myosin diluted 1:100. They were then incubated with a secondary antibody (IgG labeled with fluorescein) and counterstained with DAPI.t tests. Differences were considered statistically significant for P values < 0.05.The data are presented as mean and standard error of the mean for each group. Individual comparisons were made between groups using two-tailed Student’s Examination of fresh SIS stained with H&E, Masson’s trichrome, and DAPI Fig.  showed tScanning electron micrographs Fig.  showed dP < 0.05). The USCs grew well in medium containing the 5% PAA-treated SIS scaffold, and the OD  values in the scaffold groups were similar to those in the control group (P > 0.05)  Fig. .Single USCs were observed 3–4 days after initial seeding Fig. . These cMacroscopy showed that collagen-based scaffolds derived from porcine SIS had been mechanical and chemically treated to remove cells Fig. . ExaminaAll animals in both groups survived the duration of the study with no abnormal findings. At 3 months, macroscopy and a retrograde urethrogram of the urethra Fig.  showed eIF staining was used to detect differentiation of the seeded USCs. After labeling, USCs exhibited green fluorescence Fig. . PKH67-pP < 0.05) by histomorphometric analysis  at 3 and 4 weeks and 3 months , but the diameter of the vessels was not significantly different by histomorphometric analysis  at 3 and 4 weeks and 3 months. Additionally, all of the animals in the 3D porous SIS alone group exhibited strictures, and only one animal in the autologous USC-seeded 3D porous SIS group exhibited minor strictures. The number of vessels was significantly higher in the autologous USC-seeded 3D porous SIS group than in the 3D porous SIS alone group at early time points, which allowed host cells to infiltrate the scaffold and enhanced the transport of nutrients and metabolic waste products, thereby promoting urethral regeneration. We also demonstrated that the native urethra environment in the animal appeared to provide enough stimuli to cause the USCs to differentiate into SMCs and UCs and to assume the role of urethra SMCs and UCs. In addition, USCs secrete a variety of growth factors, such as vascular endothelial growth factor (VEGF), PDGF, PLGF, basic fibroblast growth factor, epidermal growth factor, and TGF-β [In this study, H&E and DAPI staining revealed that autologous USCs were successfully seeded into 3D porous SIS scaffolds on both sides, and the cells attached well and formed a multilayer of cells on the surface of the SIS. Additionally, H&E and AE1/AE3 IHC staining showed integrated epidermal cell layers in the autologous USC-seeded 3D porous SIS scaffold group 2 weeks after the operation. By comparison, stratified epidermal cellular layers were formed at 3 weeks. Despite the infiltration of the host epithelial cells, there was no significant difference in urothelial regeneration between the two groups at 3 months. However, the early integrated epidermal cellular layers attached to the luminal surface of the urethra may provide a permeability barrier that can prevent urine leakage from the underlying tissues, which is one of the major factors implicated in the inflammatory infiltration and fibrous tissue deposition that lead to stricture formation. Importantly, Masson’s trichrome, myosin IHC staining, and functional analysis showed that the animals that received the autologous USC-seeded 3D porous SIS scaffold showed improvement over those that received the 3D porous SIS scaffold alone. SMCs were seen in the autologous USC-seeded 3D porous SIS scaffold group 2 weeks postoperatively. These continued to develop and approximate normal tissues throughout the study with no abnormal findings. By comparison, SMCs began to appear at 3 weeks postoperatively. The smooth muscle-to-collagen ratio and the content of smooth muscle were significantly higher in the autologous USC-seeded 3D porous SIS group than in the 3D porous SIS alone group , cytokines (such as interferon and granulocyte colony-stimulating factor), and chemokines (such as macrophage inflammatory protein-1α) , and inhAlthough this study provides evidence that autologous USC-seeded 3D porous SIS scaffolds can be used to construct a tissue-engineered urethra, it has some limitations. First, we did not analyze the bioactive factors, such as growth factors and cytokines, that are secreted by USCs and which are believed to enhance tissue regeneration. Second, in the present study, the urethral defect models were established in normal animals, which cannot fully simulate the exact clinical situation of urethral strictures, which is characterized by a fibrotic urethral bed. Finally, the number of experimental animals that were evaluated per time point is small. Further investigations are necessary to translate this technology into the clinic.USCs that can be easily isolated from voided urine can be extensively expanded to high numbers in vitro, and efficiently give rise to UCs and SMCs in vitro and vivo. They can be utilized as a promising cell source for tissue-engineered urethras. In addition, 3D porous SIS as an optimized biomaterial has potential as a cell-based scaffold for tissue-engineered urethra. Furthermore, an autologous USC-seeded 3D porous SIS scaffold could promote urethral regeneration.


NPM1 and FLT3, dysregulation of microRNAs (miRNAs) is now recognized as having an important role in leukemogenesis. Distinct miRNA expression profiles can classify AML subgroups and miRNAs have recently emerged as novel therapeutic targets in hematopoietic malignancies.2Hematopoiesis depends on a tightly controlled balance of self-renewal, proliferation, cell death and differentiation. Although the disturbance of this equilibrium creates a predisposition to leukemogenesis, targeted manipulation or modulation can in turn lead to therapeutic advances. In addition to chromosomal aberrations and frequently mutated genes such as 3  was observed for patients with miR-139-5p levels above the median independent of cytogenetic subgroup  compared with the total AML population.  (P=0.001) translocation, a prognostically favorable AML subgroup  .in vivo compared with the pre-leukemic Hoxa9 cells that lead to a long latency AML.8 Upregulation of HOXA9 and MEIS1 is frequently observed in NPM1-mutated CN-AML and enhances FLT3 signaling.10 Based on the expression pattern of miR-139-5p in AML patients, we further hypothesized that miR-139-5p would be downregulated in Hoxa9/Meis1 cells compared with untransformed wild-type bm cells. Indeed, miR-139-5p was significantly downregulated in Hoxa9/Meis1 cells compared with untransduced bm cells as measured by qRT-PCR  were FACS-sorted for GFP, followed by transplantation of 2 × 105 cells per arm into lethally irradiated recipient mice.10 Restoration of miR-139 levels significantly delayed Hoxa9/Meis1-mediated leukemogenesis , which causes a rapid AML  qRT-PCR , highliget al.12 demonstrated that Hoxa9/Meis1 cells harbor three, immunophenotypically distinct compartments with varying tumor-initiating activity. Therefore, bm cells of deceased mice were analyzed by flow cytometry for their respective immunophenotype  frequency  and granulocyte-macrophage colony-stimulating factor (GM-CSF), resulting in a block of neutrophil, granulocytic and macrophage differentiation.13 Our findings suggest that miR-139-5p overexpression might partly overcome this block of differentiation in Hoxa9/Meis1 AML cells in vivo. In order to explore the potential of miR-139-5p as an inducer of myeloid differentiation, we retrovirally overexpressed miR-139-5p in 32D cells, an established model of granulocytic differentiation. Increased miR-139 levels promoted G-CSF-induced granulocytic differentiation in 32D cells , which causes a predisposition for AML.14Recently, Gibbs henotype . We foun P=0.01,  as shown2D cells , support1 restoring miR-139-5p levels with a miRNA mimic, for example in combination with G-CSF, could enhance the effect of polychemotherapy especially in the treatment of CN-AML patients, which is currently based on cytarabine and anthracyclines.On the basis of increasing interest in miRNAs as therapeutic targets in AML,14 We show for the first time that miR-139-5p is specifically downregulated in CN-AML with mutated FLT3 and acts as a strong tumor suppressor in a primary AML transplantation model. Our findings further rationalize the development of a miR-139 mimic as a treatment option for AML.The potent tumor suppressor effect of miR-139 in hematopoietic malignancies has largely gone unnoticed, given that there are only two reports so far linking miR-139 to AML.


Overall, these results have led Brooks3 to conclude that the ‘bystander effects suggest that organs respond as a unit and are not just a bag of individual cells acting independently’.The bystander effect is part of a cell’s cell–cell communication repertoire, and refers to the observation that biological effects can be observed in cells that are not directly targeted. This type of communication can be mediated by intercellular communication via gap junctions (GJs), or by messengers released from targeted cells that travel across the plasma membrane or are secreted extracellularly.4 have studied bystander effects in networks of retinal pigment epithelium (RPE) cells, using high-resolution imaging. The RPE lends itself to this kind of two-dimensional analysis, since it is composed of a single layer of hexagonal cells that are connected by tight and adherence junctions to establish barrier function, and GJs to mediate intercellular communication,5 features that can be replicated in vitro. The RPE is of interest in diseases such as age-related macular degeneration (AMD), macular edema and diabetic retinopathy, whose disease processes involve oxidative stress. The authors argued that since the RPE is a highly coupled network, any individual cell will be significantly affected by the behavior of its neighbors; and second, they suggested that the susceptibility of a given cell to bystander signals is dependent upon its prior metabolic history and mitochondrial health.Ishii and Rohrer6 it induces reactive oxygen species (ROS) production and lipid peroxidation in RPE cells7 resulting in apoptotic cell death;8 and it can cause mitochondrial damage.9 As cellular readouts of cell–cell communication and mitochondrial health, the characteristics of induction and transfer of ROS and calcium ions (Ca2+) to connected neighboring cells and their mitochondrial membrane potential (ψm) was examined  resulted in the rapid generation of a continuous ROS signal (hydrogen peroxide (H2O2), H2DCFDA) followed by the generation of hydroxyl radicals (CellRox-Green) in the stimulated cell. This was followed by a transient increase first in cytosolic and subsequently mitochondrial Ca2+ (Fluo8). Upon transfer of Ca2+ into the mitochondria, mitochondrial membrane potential changes ) were triggered, starting with a transient increase in membrane hyperpolarization followed by membrane depolarization. On the basis of the known biology of oxidative stress, these changes were predictable. However, what makes this manuscript exciting and novel, is the analysis of the bystander effect in connected cells.Stimulation of a single, randomly selected RPE cell within an established network with blue light  or 1-octanol were added. While the transfer of the ROS signal was not inhibited by GJ blockers, the transfer of the Ca2+ signal and the subsequent changes in ψm were completely eliminated.To determine the potential mechanism of signal transfer, GJ blockers 182+uptake and release, and a link between Ca2+ dysregulation, mitochondrial alterations and cell death has been evident in many disorders. Also, melanin, a hallmark of RPE, binds Ca2+. In the current study, Ca2+ levels were found not to be uniform in the resting RPE network. Interestingly, both the ROS increase and the ψm amplitude elicited by the bystander effect in a given surrounding cell exhibited a linear correlation with the cell’s baseline Ca2+ concentration. Likewise, Ca2+ levels were found to be negatively correlated with the amount of pigmentation present in a cell, supporting a role of melanin in the regulation of Ca2+ homeostasis.10 Since Ca2+ homeostasis is essential for cell survival, the RPE networks were analyzed over a 20 h period to examine the long-term consequences of photo-oxidative stress. Laser stimulation of a single RPE cells was found to induce cell death (To-Pro3) in approximately half the cells present in the recording window. Application of βGA reduced cell death to ~10%. Intriguingly, and similar to the short-term results on mitochondrial health, the higher the Ca2+concentration at baseline, the greater the risk for the cell to undergo future cell death.Calcium is an essential intracellular signaling molecule, and its levels are tightly regulated. Relevant for this study, mitochondria and the endoplasmic reticulum (ER) have important roles in Ca2+-storing organelles, and mitochondria as the major checkpoint for apoptosis have been studied extensively. Ca2+ transfer between the cytoplasm and the ER is mediated via the sarco/ER Ca2+-ATPase (SERCA), its ER release is mediated via the activation of either IP3 or ryanodine receptors, and mitochondrial uptake is accomplished via a Ca2+-uniporter.11 In the RPE network, a critical role for the ER could be identified, as the SERCA inhibitor thapsigargin (Sigma Aldrich) reduced cell death to <10%. ER Ca2+ release was mediated by ryanodine receptors, as the IP3-receptor antagonist – 2-aminoethoxydiphenyl borate (2APB) did not inhibit cell death, whereas the ryanodine-receptor antagonist, dantrolene did. Finally, an essential role for mitochondrial Ca2+ release was verified by demonstrating that treatment with the mitochondrial Ca2+ uptake inhibitor Ru360 completely inhibited cell death. Together, these results suggest that Ca2+-mediated cell death in RPE networks is mediated by ER-mitochondria Ca2+ transfer.11The ER and mitochondria as Ca4 convincingly demonstrate that local photo-oxidative stress in a donor RPE cell can trigger cellular damage that require a dual-hit of ROS and Ca2+-related signals. The identified metabolic signature of elevated baseline Ca2+ levels should be further investigated as a contributing factor to disease processes in networks of cells in which initial damage seems to occur in susceptible, and is delayed in more resilient areas.The results presented by Ishii and Rohrer


Here we find that extracts from dust mite and cockroach induce sustained Ca2+ elevations in AECs through the activation of Ca2+ release-activated Ca2+ (CRAC) channels encoded by Orai1 and STIM1. CRAC channel activation occurs, at least in part, through allergen mediated stimulation of PAR2 receptors. The ensuing Ca2+ entry then activates NFAT/calcineurin signaling to induce transcriptional production of the proinflammatory cytokines IL-6 and IL-8. These findings highlight a key role for CRAC channels as regulators of allergen induced inflammatory responses in the airway.Aberrant immune responses to environmental allergens including insect allergens from house dust mites and cockroaches contribute to allergic inflammatory diseases such as asthma in susceptible individuals. Airway epithelial cells (AECs) play a critical role in this process by sensing the proteolytic activity of allergens via protease-activated receptors (PAR2) to initiate inflammatory and immune responses in the airway. Elevation of cytosolic Ca Asthma is a heterogeneous disease arising from a complex interplay of genetic, epigenetic and environmental factors and is believed to affect over 300 million people worldwide1311Alternaria have been implicated in the release of IL-6, GM-CSF and IL-33 from AECs891011122+ elevations in AECs, which in principle could drive transcriptional and enzymatic cascades to induce cytokine production912132+ fluxes arise primarily due to Ca2+ release from internal stores or if they also involve activation of specific Ca2+ channels on the plasma membrane. Given recent evidence implicating Ca2+ signaling in multiple effector functions in AECs152+ signals in response to allergens is needed to better understand how cytokine signaling pathways are induced in AECs and to translate this knowledge in the quest for identifying novel targets for therapy.Among the numerous immune and structural cells implicated in the allergen response, airway epithelial cells (AECs) are thought to play an early role in the process through direct interactions with allergens. Located at the interface of the host and the environment, AECs act not only as a mechanical barrier and a first line of defense against inhaled allergens, but also play a vital role in their early recognition and response using pattern recognition receptors such as toll-like receptors (TLRs) and PARs632+ release-activated Ca2+ (CRAC) channels are the primary mechanism for producing cytosolic Ca2+ elevations in many non-excitable cellsOrai genes (Orai1-3) and activated by the ER Ca2+ sensors, STIM1-21718192+]ER, and, in response to ER Ca2+ store-depletion, translocate to the junctional ER to physically interact with Orai channels resulting in CRAC channel gating172+ influx regulates a variety of functions including gene expression, cell proliferation and differentiation in many types of cells17Store-operated Ca2+ signals in AECs and are activated in response to PAR2 stimulation152+ elevation regulates the production of key inflammatory cytokines including IL-6, IL-8, and TSLP2+ elevations in AECs and are thought to mediate their inflammatory effects, at least in part, by stimulating PAR2 receptors91112911122+ signal induces the generation of IL-6 and IL-8. These results highlight an important role for CRAC channels as key regulators for early activation of allergic inflammation in AECs.We and others have shown that CRAC channels are a major mechanism for eliciting CaAlternaria, Aspergillus) induce cytosolic Ca2+ signals in AECs8132+ signals, yet it is not clear whether they primarily arise due to Ca2+ release from internal stores or if they additionally involve Ca2+ influx across the plasma membrane. We and others have previously shown that store-operated calcium entry (SOCE) is a major mechanism of Ca2+ influx in bronchial epithelial cells and is stimulated by activation of PAR2 receptors152+ imaging  and fungi  was unaffected following prior treatment with the allergen. Likewise, pre-application of the PAR2 agonist trypsin impaired a subsequent Ca2+ response to cockroach allergen but not to UTP.  allergens and the airway epithelium underlie the development of airway inflammation seen in allergic diseases like asthma362+, which has been proposed to occur through both PAR2 dependent and independent pathways1011122+ entry pathways that mediate allergen-evoked Ca2+ signals have not been determined. In this study, we show that insect allergens derived from cockroach and dust mite extracts activate CRAC channels in bronchial epithelial cells. CRAC channel activation occurs, at least in part, through stimulation of PAR2 receptors and mediates an important role in the induction of the inflammatory modulators IL-6 and IL-8.Interactions between common allergens found in ambient air such as insect  or fungal  will help to discern the contributions of precise components of the dust mite extracts to the observed Ca2+ signals.Several studies have established a role for PAR2 receptors in the induction of Ca protein . However2+ elevation evoked by extracts of the Alternaria fungus. A previous study that used a high concentration of Alternaria extracts (200 μg/mL) found that the extracts cause Ca2+ elevations acting through the autocrine stimulation of purinergic receptors by ATP2+ elevations in the presence of extracellular Ca2+, no response occurred in Ca2+-free Ringer’s buffer, arguing against activation of purinergic receptors, at least in the concentration range we tested. Moreover, the observed Ca2+ influx seen in the presence of extracellular Ca2+ was not dependent on CRAC channels as the CRAC channel inhibitor BTP2 had no effect. These results indicate that Alternaria evokes Ca2+ influx through other Ca2+ influx pathways whose identity remains to be determined.Interestingly, we failed to detect the involvement of CRAC channels in the Ca2+ signal is known to play an important role in the induction of the cytokines IL-6 and IL-8 through an NFAT dependent mechanism. These results demonstrate that CRAC channels may have a central role as effectors of allergen signaling in the airway epithelium.In conclusion, we provide evidence showing that bronchial epithelial cells sense cockroach and dust mite allergens through the activation of cell surface PAR2 receptors, which in turn leads to the opening of store-operated CRAC channels. The ensuing Ca2. Cells from passage 44-51 were used for experiments.The bronchial epithelial cell line BEAS-2B, a kind gift from Curtis C. Harris , was cultured in DMEM/F12 medium (CellGro) containing 5%FBS (Hyclone), 50 U/mL penicillin and 50 mg/mL Streptomycin and was maintained at 37 °C and in 5% COsiRNAs used to down regulate STIM1, STIM2, Orai1 and PAR2 protein expression along with scrambled siRNA negative control were purchased from Ambion, Life Technologies (SilencerSelect predesigned siRNA). siRNA constructs were transfected into BEAS-2B cells using Lipofectamine2000 (Invitrogen) according to manufacturer instructions. Cells were used for experiments 48–72 hours after transfection.2, 2 CaCl2 and 5 Na-HEPES. pH was adjusted to 7.4 using NaOH. For the Ca2+ free Ringers solution, CaCl2 was excluded from the above composition and MgCl2 was increased to 3 mM. Stock solutions of BTP2, thapsigargin (TG) and cyclosporinA (CsA) were made in DMSO. PAR2-agonist type IX trypsin (Sigma) was constituted in water. Dust mite allergen extract (nDer p) was from Indoor Biotechnologies Inc, Charlottesville, VA. Cockroach extract was purchased from HollisterStier Allergy, Spokane, WA. CRAC channel inhibitor BTP2 was from Millipore, Billerica, MA. All other compounds were from Sigma Aldrich, St. Louis, MO.The standard extracellular Ringers solution had the following composition (in mM): 150 NaCl, 4.5 KCl, 10 D-glucose, 1 MgCl2+]i measurements were done according to the protocol described previouslyF340/F380 intensity ratio was obtained as a function of time. The ratios were converted to [Ca2+]I using the formulaBEAS-2B cells were grown on poly-L lysine coated glass bottom dishes . Cells were loaded with 2.5 μM Fura-2 AM  in DMEM/F12 and 5% FBS culture media for 40 minutes at room temperature. Excess fura2 was washed off and cells were incubated in media for an additional 10 minutes before imaging. Single cell [CaF340/F380 fluoresce intensity ratio and Rmax (9.645) and Rmin (0.268) were determined by in-vitro calibration of FURA2Fmin/Fmax ratio at 380 nm and Kd is the apparent dissociation constant of fura-2 binding to Ca2+ (135 nM).where R is the BEAS-2B cells were cultured in 6-well plates. At 70% confluency, cells were washed with cold PBS and lysed in a solution containing 150 mM NaCl, 50 mM Tris, 1% Triton-X-100, 0.1% SDS and 1x Protease Inhibitor Cocktail (Sigma) for 45 minutes. Cell lysates were obtained using a cell scraper, lysates were spun down at 4 °C for 30 minutes and supernatants were collected and stored at −80 °C. For Western blotting, samples were heated to 99 °C for 5 minutes in Laemmli Sample Buffer (Bio-Rad) containing 0.1% β-mercaptoethanol, run on 10% SDS-PAGE gels, and transferred to nitrocellulose membrane. Orai1, STIM1, and STIM2 proteins were detected using an affinity purified polyclonal antibodies and peroxidase labelled secondary antibodies43BEAS-2B cells were cultured on 24 well plates in DMEM/F12 media with 5% FBS. 24–48 hours later, cells were pre-treated with CRAC channel inhibitor BTP2 (500 nM) or calcineurin inhibitor Cyclosporin A (500 nM) for 45–60 min before being stimulated with dust mite and cockroach allergens for 6 or 24 hours. Supernatants were collected and stored at −80 deg. C. Levels of inflammatory mediators IL-6 and IL-8 was measured using ELISA kits .2+ traces and bar graphs summarizing the data are reported as mean ± SEM. For data sets involving more than two groups, initial statistical analysis was performed using ANOVA with a confidence interval of 5%. This was followed by two-tailed paired student t-test for comparing different treatment conditions within the set.Average cytosolic CaHow to cite this article: Jairaman, A. et al. Allergens stimulate store-operated calcium entry and cytokine production in airway epithelial cells. Sci. Rep.6, 32311; doi: 10.1038/srep32311 (2016).


Acute biliary pancreatitis is a sudden and severe condition initiated by bile reflux into the pancreas.2+ signals and necrosis in isolated pancreatic acinar cells but the effects of bile acids on stellate cells are unexplored.Bile acids are known to induce Ca2+ signals and necrosis in stellate cells compared to the adjacent acinar cells in pancreatic lobules; whereas taurolithocholic acid 3‐sulfate primarily affects acinar cells.Here we show that cholate and taurocholate elicit more dramatic Ca2+ signals and necrosis are strongly dependent on extracellular Ca2+ as well as Na+; and Na+‐dependent transport plays an important role in the overall bile acid uptake in pancreatic stellate cells.CaBile acid‐mediated pancreatic damage can be further escalated by bradykinin‐induced signals in stellate cells and thus killing of stellate cells by bile acids might have important implications in acute biliary pancreatitis.2+ signals and necrosis in acinar cells. However, bile acid‐elicited signalling events in stellate cells remain unexplored. This is the first study to demonstrate the pathophysiological effects of bile acids on stellate cells in two experimental models: ex vivo (mouse pancreatic lobules) and in vitro (human cells). Sodium cholate and taurocholate induced cytosolic Ca2+ elevations in stellate cells, larger than those elicited simultaneously in the neighbouring acinar cells. In contrast, taurolithocholic acid 3‐sulfate (TLC‐S), known to induce Ca2+ oscillations in acinar cells, had only minor effects on stellate cells in lobules. The dependence of the Ca2+ signals on extracellular Na+ and the presence of sodium–taurocholate cotransporting polypeptide (NTCP) indicate a Na+‐dependent bile acid uptake mechanism in stellate cells. Bile acid treatment caused necrosis predominantly in stellate cells, which was abolished by removal of extracellular Ca2+ and significantly reduced in the absence of Na+, showing that bile‐dependent cell death was a downstream event of Ca2+ signals. Finally, combined application of TLC‐S and the inflammatory mediator bradykinin caused more extensive necrosis in both stellate and acinar cells than TLC‐S alone. Our findings shed new light on the mechanism by which bile acids promote pancreatic pathology. This involves not only signalling in acinar cells but also in stellate cells.Acute biliary pancreatitis, caused by bile reflux into the pancreas, is a serious condition characterised by premature activation of digestive enzymes within acinar cells, followed by necrosis and inflammation. Bile acids are known to induce pathological Ca Acute biliary pancreatitis is a sudden and severe condition initiated by bile reflux into the pancreas.2+ signals and necrosis in isolated pancreatic acinar cells but the effects of bile acids on stellate cells are unexplored.Bile acids are known to induce Ca2+ signals and necrosis in stellate cells compared to the adjacent acinar cells in pancreatic lobules; whereas taurolithocholic acid 3‐sulfate primarily affects acinar cells.Here we show that cholate and taurocholate elicit more dramatic Ca2+ signals and necrosis are strongly dependent on extracellular Ca2+ as well as Na+; and Na+‐dependent transport plays an important role in the overall bile acid uptake in pancreatic stellate cells.CaBile acid‐mediated pancreatic damage can be further escalated by bradykinin‐induced signals in stellate cells and thus killing of stellate cells by bile acids might have important implications in acute biliary pancreatitis. Acute pancreatitis (AP) is a potentially severe disease with an overall mortality up to 6% , especially in the context of pancreatic enzyme secretion, which, in physiological conditions, is induced by acetylcholine (ACh) or cholecystokinin (CCK) and regulated by intracellular Caet al. et al. et al. et al. et al. et al. et al. et al. et al. et al. et al. et al. et al. Although cells relevant to exocrine and endocrine functions of the pancreas have been extensively studied and well described in the literature  and detailed signalling events in PSCs during development of pancreatitis remain largely unexplored. Evidence shows that bile acids may act on other cell types in the pancreas, such as duct cells, by inducing pathological Ca2+ signals and affecting ductal secretion  were supplied by Charles River, maintained on a standard rodent chow diet with free access to water, and housed in the institutional animal unit (12 h light cycle). The mice were killed according to Schedule 1 of Animals (Scientific Procedures) Act 1986, dissected and the pancreatic tissue was removed for further experimental procedures. In order to reduce the number of animals used in the study, where applicable, some experiments were performed on cells cultured m): NaCl 140, KCl 4.7, Hepes 10, MgCl2 1, glucose 10; pH 7.3. NMDG‐Hepes was a modification of NaHepes, where NaCl was replaced by 140 mmN‐methyl‐d‐glucamine (NMDG+), pH 7.3.The main reagents for cell isolation and microscopy include: Fluo‐4 AM, propidium iodide and Hoechst‐33342 ; collagenase type V, inorganic salts and bile salts : sodium cholate (NaChol), sodium taurocholate (TC) and taurolithocholic acid 3‐sulfate (TLC‐S). NaHepes buffer was prepared as follows  and subsequently incubated at 37°C for 5–6 min in the collagenase solution to allow for partial digestion of the tissue. After incubation the pancreas was broken down by pipetting, suspended in NaHepes, spun , resuspended in NaHepes and spun again. Finally, isolated pancreatic lobules were suspended in NaHepes and loaded with Fluo‐4 AM as described below.Lobule preparation and most of the experimental work was carried out in NaHepes buffer. Unless otherwise stated, NaHepes was supplemented with 1 m2 and split once a week. A frozen stock of hPSC was prepared after the first passage and was used to revive the culture every 5–6 weeks.Human pancreatic stellate cells (hPSCs) and stellate cell complete medium (SteCM) were obtained from ScienCell, Carlsbad, CA, USA. hPSCs were cultured in SteCM in T25 flasks at 37°C, 5% COm Fluo‐4 AM  for 1 h at 30°C. After the incubation pancreatic lobules were resuspended in fresh NaHepes and used for experiments at room temperature in a flow chamber perfused with NaHepes‐based extracellular solution. hPSCs were plated on sterile round coverslips and loaded with 1 μm Fluo‐4 AM for 30 min at 37°C. After the incubation the glass coverslips with hPSCs were used for the flow chamber assembly. Ca2+ imaging was performed using Leica confocal microscope TCS SPE with a ×63 oil objective. Fluo‐4 AM was excited with a 488 nm laser at 1–3% power and emitted light was collected at 500–600 nm. Static cell images were taken at 512 × 512 pixel resolution and series of images were recorded at 256 × 256, two consecutive frames were averaged, and time resolution was 1 image per 2 s. Fluorescence signals were plotted as F/F0, where F0 was an averaged signal from the first ten baseline images. For very long experiments linear correction of focus drift was applied.Isolated mouse pancreatic lobules were loaded with 10 μ2+). Ten minutes before the end of the incubation, perfusion was stopped and propidium iodide (PI) was added (2 μg ml−1).For experiments with 30 min incubation pancreatic lobules were isolated and loaded with Fluo‐4 AM. Then the cells were perfused in a flow chamber with NaHepes‐ or NMDG‐Hepes‐based solution containing bile acid salts (with or without Cam Fluo‐4 was added and the incubation continued for another hour. 15 min before the end of the incubation PI (1 μg ml−1) and Hoechst‐33342 (5 μg ml−1) were added. Cells were visualised with Leica confocal microscope TCS SPE. Fluo‐4 fluorescence allowed for detection of PSCs in pancreatic lobules, PI specifically stained necrotic cells  and Hoechst marked all nuclei  making possible calculation of the total number of cells. Pancreatic lobules were imaged by collecting multiple pictures along z axis, 5 μm apart. Then the pictures were merged yielding a maximum projection image, where live and necrotic cells were counted. Five to ten series were collected per sample.In experiments involving long incubations (2 h) pancreatic lobules were kept in NaHepes or NMDG‐Hepes in the presence of bile acid. After the first hour, 10 μ2. Then the medium was replaced by NaHepes or NMDG‐Hepes containing 0.1 or 1 mm of NaChol or TC; and hPSCs were incubated for 2 h at 37°C. Fifteen minutes before the end of the incubation PI (1 μg ml−1) and Hoechst‐33342 (5 μg ml−1) were added. PI stained necrotic cells and Hoechst stained nuclei. Multiple pictures (15–20) per treatment group were taken; live, apoptotic and necrotic cells were counted in each treatment group.hPSCs were plated on 35 mm glass bottom microwell dishes  and grown for 24 h in SteCM at 37°C, 5% COslc10A1 (CGT CCT CAA ATC CAA ACG GC/ACT TCA GGT GGA AAG GCC AC), human desmin (GAT CCA GTC CTA CAC CTG CG/CTC GGA AGT TGA GGG CAG AG), bdkrb1 (TGG GAC CAC AGG TCA CTG/CCA GGT TGG CCA GGT AGA TT), bdkrb2 (CTG TTC GTG AGG ACT CCG TG/AGG TAG ATC TCT GCC ACC GT), slco4A1 (ATC TAC ACG GAA ATG GGC CG/ACA TGC CGG TGA TGA GAG TG), slco1B3 (TGG CTT GGT TTC CTT GTG TC/CCA GTT GCA ACC GTA GGA AT) and α‐sma (TTC CAG CCA TCC TTC ATC GG / CCC GGC TTC ATC GTA TTC CT). The PCR reaction conditions were as follows: 5 min 95°C; then 40 cycles of 30 s 95°C, 1 min 56°C, 1 min 72°C; and finally 5 min 72°C. The PCR products were resolved on 1% agarose gel with Gel Red Nucleic Acid Gel Stain .Total RNA was extracted from hPSCs using the PureLink RNA Mini Kit (ThermoFisher Scientific). Human hepatocyte cDNA was obtained from ScienCell. Reverse transcription was performed with the GoScript Reverse Transcription System . cDNA was amplified using GoTaq G2 DNA Polymerase (Promega) and specific gene primer pairs (given in forward/reverse order) for 2O); then 4 μl of sample reducing agent and 10 μl 4 × NuPage LDS sample buffer were added. Samples were heated at 95°C for 10 min and then spun . Proteins were separated by SDS‐PAGE , then transferred on nitrocellulose membrane 0.45 μm , followed by 1 h incubation in 5% milk in Tris‐buffered saline with 0.1% Tween 20 . Primary antibody anti‐NTCP  was prepared in a 1:2000 dilution in TBS‐T with 1% milk and incubated for 1 h. Membranes were then washed (3 × 10 min in TBS‐T) and incubated for 1 h in a 1:5000 dilution of HRP‐conjugated goat anti‐rabbit IgG secondary antibody  in TBS‐T with 1% milk. The membranes were washed (4 × 10 min in TBS‐T) and then visualised using ImmunoCruz Western Blotting Luminol Reagent (Santa Cruz Biotechnology) and ChemiDoc XRS+ Imaging System (Bio‐Rad).Unless otherwise stated all reagents for immunoblotting mentioned below were obtained from ThermoFisher Scientific. Total protein was isolated from hPSCs, mouse pancreas, liver, kidney, spleen, lungs and suspended in RIPA buffer (Sigma‐Aldrich). Human liver tissue lysate was obtained from Novus Biologicals, Littleton, CO, USA. Protein concentration was determined using DC Protein Assay . For each sample the volume containing 75 μg of protein was brought to 26 μl by addition of double‐distilled water . Antigen retrieval was performed in TAE buffer (pH 8.1) in an autoclave  and then the sections were allowed to cool at room temperature (30 min) before permeabilisation in 0.4% Triton X‐100 in ddH2O (10 min). Then the sections were washed 3 × 5 min in 0.1% Tween 20 in ddH2O (washing solution). In order to quench autofluorescence the sections were incubated in 0.2% Sudan Black B for 20 min , initially for 1 h at room temperature, followed by an overnight incubation at 4°C inside a humid chamber. Negative controls were incubated in the blocking solution without antibody. The following day the sections were washed in the washing solution (4 × 5 min), and incubated for 1 h at room temperature with the appropriate secondary antibody (4 μg ml−1 or 1:500 in the blocking buffer). After the incubation the sections were washed in the washing solution (4 × 5 min), embedded in ProLong Diamond Antifade Mountant with DAPI (ThermoFisher Scientific) and imaged immediately using Leica sp5 confocal microscope. The slides were then stored at 4°C.Formalin‐fixed paraffin‐embedded tissue sections (4 μm) were heated in a dry oven , and then deparaffinised in xylene (2 × 10 min). The sections were rehydrated by subsequent washes in ethanol solutions with increasing content of ddHet al. Primary antibodies: rabbit polyclonal anti‐NTCP antibody   recorded between 200 and 2000 s were calculated and then averaged and presented as bar charts with standard errors. The Student's t test was applied for statistical comparison. The significance threshold was set at 0.05. Where applicable, N indicates the number of individual experiments/cell isolations, whereas n indicates individual cells.For cell death assays, three to six independent experiments were performed for each treatment group on cells isolated from different animals; average values and standard errors of the mean were calculated and results presented as bar charts. Statistical analysis was performed using the Student's ex vivo model, closely resembling the native environment of the pancreas, which allows for investigation of signalling events simultaneously induced in different cell types. Figure A shows staining of a pancreatic lobule with the Ca2+‐sensitive dye Fluo‐4 AM, where PSCs exhibit a stronger signal as compared to the surrounding PACs. ATP at micromolar concentrations and bradykinin (BK) at low nanomolar concentrations are known to induce elevations in the cytosolic [Ca2+] ([Ca2+]i) of PSCs . Figure B and C demonstrate typical [Ca2+]i elevations recorded in PSCs upon stimulation with 10 nm BK and 2 μm ATP, respectively. BK consistently induced biphasic responses: an initial large cytosolic Ca2+ transient followed by a sustained Ca2+ plateau caused by Ca2+ entry . Since BK induces Ca2+ rises in PSCs but not in PACs, acute Ca2+ signal generation in response to BK was frequently used in this study to verify the stellate phenotype in cell clusters.Although the exocrine pancreas mainly consists of pancreatic acinar cells (PACs), other less conspicuous cell types, such as pancreatic stellate cells (PSCs), are woven into the tissue. Two experimental models were used in this study: 1) pancreatic lobules isolated from the mouse pancreas; (2) and primary pancreatic stellate cells of human origin (hPSCs). Pancreatic lobules are a perfect  pancreat2+‐sensitive dye Fluo‐4 AM are depicted in Fig. D. BK at 1 μm, but not 10 nm, induced a single cytosolic Ca2+ transient without a sustained plateau phase , which is similar to what has previously been shown for PSCs in culture .hPSCs stained with the Ca2+]i elevations via intracellular store depletion and subsequent Ca2+ entry . TC (5 mm) also caused Ca2+ signals in PSCs and only modest oscillations in PACs . In contrast, TLC‐S at micromolar concentrations has already been demonstrated to trigger [Ca2+]i elevations and cell death in PACs and to activate Ca2+‐independent cationic currents , inducing robust Ca2+ oscillations in PACs, but not in PSCs. The latter showed typical responses to 10 nm BK.Here, we tested the effects of three bile acid salts, sodium cholate (NaChol), taurocholate (TC), and taurolithocholic acid 3‐sulfate (TLC‐S), on Fluo‐4‐loaded pancreatic lobules. Figure  small Ca+i oscill small Ca+i oscill small Ca+i oscillm and 5 mm NaChol caused rapid and global [Ca2+]i elevations in hPSCs, whereas 100 nm BK induced only very modest oscillations . Treatment with 100 μm NaChol or TC, a ten times lower concentration than the lowest concentration capable of triggering at least a minor [Ca2+]i elevation in pancreatic lobules, was sufficient for induction of substantial [Ca2+]i elevations in hPSCs .Similarly, 1 mglobal Ca+i elevatglobal Ca+i elevat2+ entry in PACs  or 5 mm TC  in the absence of extracellular Ca2+. Apart from small oscillations  bile acids consistently failed to induce marked increases of [Ca2+]i in either cell type. Upon readmission of extracellular Ca2+ (1 mm), PSCs developed global [Ca2+]i elevations, whereas only very modest oscillations were seen in PACs . Readmission of Ca2+ to untreated cells did not cause any responses . Similarly, removal of extracellular Ca2+ blocked responses to 1 mm NaChol  and 1 mm TC  in hPSCs and readmission of extracellular Ca2+ recovered the responses.Previous reports have demonstrated that certain bile acids empty intracellular stores triggering Cahol Fig. A or 5 m TC Fig. B in thehol Fig. E and 1  TC Fig. F in hPS2+ elevations, particularly since PSCs did not develop global Ca2+ responses to 5 mm NaChol in the absence of external Ca2+. However, at an extracellular [Ca2+] of 100 μm, which does not efficiently support Ca2+ influx via store‐operated channels . Furthermore, Fig. D shows that Ca2+ signals elicited by 5 mm NaChol in PSCs were completely inhibited by 1 mm Gd3+. Gd3+ also blocked responses in hPSCs to 100 μm NaChol . In the presence of Gd3+, an increase in the NaChol concentration to 1 mm only resulted in short‐lasting Ca2+ oscillations followed by a quick return to the resting level . Since Gd3+ is a non‐specific, but very potent, blocker of Ca2+ channels  and ryanodine receptors (RyRs) . Upon removal of caffeine, NaChol‐elicited signals were restored and subsequent reapplication of caffeine abolished the responses again . Application of 20 mm caffeine alone did not trigger [Ca2+]i elevation in hPSCs .In PACs, it has been shown that bile acids release Caeptors IPRs and ryeptors IPRs and ryeptors IPRs and ry2+ signals induced by bile acids in PSCs and their implications for acute pancreatitis, we performed a necrosis assay in which isolated pancreatic lobules were treated for 30 min with one of three bile acid salts: 5 mm NaChol, 5 mm TC or 200 μm TLC‐S in the presence or absence of extracellular Ca2+. Figure A shows that in the presence of extracellular Ca2+, both NaChol and TC caused substantial necrosis in PSCs  as compared to control . In the absence of extracellular Ca2+ the levels of NaChol‐ and TC‐elicited necrosis were markedly reduced . Neither NaChol nor TC induced substantial necrosis in PACs in the time frame of 30 min, regardless of whether extracellular Ca2+ was present or not . In contrast, TLC‐S, in the presence of Ca2+, induced necrosis in PACs (24.2 ± 2.2%); and Ca2+ removal led to a reduction of TLC‐S‐elicited necrosis in these cells . Figure B shows representative images of pancreatic lobules treated with NaChol in the presence (top panel) and absence (bottom panel) of extracellular Ca2+. PI‐stained necrotic PSCs are seen as bright red circle spots only in the top panel. These results demonstrate that PSC necrosis was a direct downstream effect of pathological Ca2+ signals caused by the bile acid salts NaChol and TC. Inhibition of these signals by removal of extracellular Ca2+ abolished cell death in PSCs. Further, TLC‐S, which did not trigger Ca2+ signals in mouse PSCs, failed to induce necrosis in these cells.In order to investigate the pathophysiological consequences of Ca+‐dependent or ‐independent mechanisms  instead of Na+ (NMDG‐Hepes). Since cholate and taurocholate were applied as sodium salts, the removal of external Na+ was not complete, but the extracellular Na+ concentration was effectively reduced from 140 mm to 5 mm. In PSCs, replacement of extracellular Na+ with NMDG+ reduced very markedly the magnitude of the [Ca2+]i elevations elicited by NaChol  or TC , whereas in PACs the tiny Ca2+ oscillations evoked by these bile acids were similar in the presence and absence of extracellular Na+ . In order to quantitatively compare the responses, the area between 200 and 2000 s under each individual trace was calculated and the average values were compared in Fig. C. In the absence of extracellular Na+, the response areas calculated for NaChol‐ or TC‐treated PSCs were significantly smaller than those of PSCs in normal NaHepes .Cellular uptake of bile acids is generally facilitated by specific membrane transporters utilising either NaD shows the extent of cell necrosis induced by 30 min incubation of pancreatic lobules with 5 mm NaChol or 5 mm TC in NMDG‐Hepes and compares these necrosis levels with those induced in normal NaHepes . Even though the absence of Na+ was associated with slightly higher baseline levels of necrosis in PSCs (P = 0.011), there was a substantial decrease in the degree of PSC necrosis induced by NaChol  or TC  in NMDG‐Hepes compared to the same concentration of the bile acid in NaHepes. PAC necrosis remained at the same level in all tested samples.Figure 2+ signals induced by 100 μm NaChol  or 100 μm TC , were abolished when NaHepes was replaced by NMDG‐Hepes. This is reflected by a significant difference in average areas calculated between 200 and 2000 s under individual traces . Figures G and H show cell death in hPSCs exposed for 2 h to bile acid salts in the presence or absence of extracellular Na+. Treatment with 0.1 mm NaChol triggered low levels of necrosis (13.4 ± 3.5%), which was abolished by substitution of Na+ with NMDG+ . At the higher concentration of 1 mm, NaChol induced significantly more necrosis in NaHepes buffer (68.6 ± 6.8%) than in NMDG‐Hepes . TC induced comparable levels of necrosis at 0.1 and 1 mm, when applied in NaHepes buffer (25.3 ± 5.3% and 27.3 ± 3.0%), and these effects were markedly decreased upon removal of extracellular Na+ . Taken together, these results show that PSCs not only utilise Na+‐dependent transport of bile acids, but that these mechanisms play significant roles in their pathophysiology.In hPSCs, Cahol Fig. Ea or 10 TC Fig. Fa, wereslc10A1) and two Na+‐independent transporters for comparison: OATP4A1 (slco4A1) and OATP1B3 (slco1B3). hPSCs express detectable levels of NTCP and OATP4A1, but not OATP1B3 .The effects of bile acids on PSCs could be explained by the transport characteristics and the substrate specificity of the sodium–taurocholate cotransporting polypeptide (NTCP). We therefore attempted to probe for NTCP at mRNA and protein levels in PSCs. cDNA, generated from mRNA of hPSCs and human hepatocytes, was amplified in a PCR reaction with primers designed for human NTCP . NTCP is a glycoprotein with an apparent molecular weight of 50 kDa (fully glycosylated) or 33.5 kDa (not glycosylated) . The liver tissue was used as a positive control. The upper panel in Fig. C shows that NTCP (white) is expressed in mouse PSCs as string‐like structures in between pancreatic acini. The fluorescence signal is accumulated close to the elongated nuclei of PSCs, but is not present in PACs. The lower panel shows a very specific diamond‐shaped pattern of NTCP at the basolateral domain of mouse hepatocytes, which is in line with previous reports  and the bradykinin receptor type 2 (BDKRB2) (red). The latter is present in PSCs, but not in PACs, and thus is used here as a cell type marker. The overlay image shows that the NTCP signal mostly co‐localises with BDKRB2, indicating that both proteins are expressed in PSCs (white arrows).Finally, we performed immunohistofluorescence staining (IHF) for NTCP on paraffin‐fixed sections of the mouse pancreas and liver Fig. C. The l+‐dependent transporters: apical sodium‐dependent bile acid transporter (ASBT) and sodium‐dependent organic anion transporter (SOAT). The polyclonal antibody used for immunoblotting was designed to bind a fragment in the middle region of human NTCP (141–154 amino acids): YSRGIYDGDLKDKV. Figure E shows a comparison of the immunogen to the homological sequences of Na+‐dependent transporters belonging to the SLC10 family in mouse. Only mouse NTCP shows high sequence identity (93%) to the immunogen, whereas ASBT and SOAT do not, and thus it is unlikely that they serve as epitopes for the antibody. The polyclonal antibody used for IHF was generated with a 14‐amino acid peptide GTHNGNIPPLQPGP, corresponding to the C‐terminal end (339–352) of rat NTCP . It is therefore expected to specifically bind mouse NTCP, but not other members of the SLC10 family.The SLC10 protein family also contains at least two other Na2+ signals in PSCs, but not in PACs . BK is generated from kininogens by enzymatic cleavage by kallikreins, which also happen to be stored in the zymogen granules of PACs . There is no direct transfer of Ca2+ signals from PSCs to PACs , but PSCs could exert effects on PACs via different types of interaction and thus contribute to the pathogenesis of pancreatitis. Figure A shows the extent of necrotic cell death induced by 2 h treatment with 200 μm TLC‐S in the presence of 10 nm BK. BK alone neither induced necrosis in PSCs (P = 0.25) nor in PACs (P = 0.15) when compared to the control; and 200 μm TLC‐S applied alone only slightly increased necrosis in PSCs  predominantly killing PACs . However, addition of BK to the TLC‐S treatment caused significantly more necrosis both in PSCs  and in PACs  as compared to TLC‐S alone. This shows that relatively modest effects of TLC‐S on PSCs can be dramatically enhanced in the presence of BK. Intensified necrosis of PSCs could further increase PAC death.Damage to the pancreatic tissue is associated with inflammation and BK is an inflammatory mediator that, at pathophysiologically relevant concentrations, induces CaB shows that combined treatment for 1 h with 5 mm NaChol and 200 μm TLC‐S caused substantial necrosis in PSCs (72.8 ± 7.5%), similar to that elicited by NaChol alone ; and markedly increased necrosis in PACs as compared to TLC‐S alone . Inhibition of Na+‐dependent uptake of NaChol in PSCs (NMDG+) resulted in a marked reduction of NaChol‐induced necrosis in PSCs . However, while neither NaChol‐ nor TLC‐S‐induced cell death in PACs was affected by removal of Na+ , combined treatment with TLC‐S and NaChol no longer induced more necrosis in PACs than TLC‐S alone . Lack of the exacerbated necrosis in PACs in the absence of Na+ may be attributed to a much smaller proportion of adjacent PSCs killed by NaChol , as compared to the level seen in the presence of Na+ (72.8 ± 7.5%). These results suggest that necrosis induced in pancreatic lobules depends on the effects of bile acids on both PACs and PSCs. Extensive necrosis in PSCs may further sensitise PACs to bile acid‐induced killing.Bile reflux leads to exposure of the pancreas to a concentrated mixture of bile acids, not just a single component. Figure 2+ signals in PSCs followed by necrotic death, but have very little effects on the neighbouring PACs. Another bile acid, TLC‐S, acts primarily on PACs and only to a lesser extent on PSCs. Although PACs are known to respond to NaChol and TC when isolated as single cells or small clusters  but also stellate cells (PSCs) are important targets for the action of bile acids in biliary acute pancreatitis. Even though PACs and PSCs are located in close proximity in pancreatic lobules they respond independently to pathophysiological stimuli and display very different sensitivities. Some of the naturally occurring bile acid salts, NaChol and TC, elicit dramatic Ca2+ stores may therefore be smaller than those of PACs. Thus the development of large Ca2+ signals requires influx of Ca2+ from the extracellular space. Therefore, it is not surprising that removal of Ca2+ from the extracellular buffer completely abolished both Ca2+ signals and necrosis in PSCs . Since inhibition of bile acid‐induced Ca2+ signals by caffeine  indicates involvement of IP3Rs and the ER, we postulate that bile acid salts, upon entering PSCs, activate intracellular Ca2+ release through IP3Rs. A very similar mechanism was already proposed to explain TLC‐S‐induced Ca2+ signals in PACs  channels in the plasma membrane . The importance and the magnitude of Ca2+ entry in these cells is also reflected by the biphasic response to BK , where the initial short‐lasting Ca2+ transient is always followed by a prolonged elevated [Ca2+]i plateau caused by Ca2+ entry .PSCs are different from PACs in many respects. They are relatively small with a big centrally located nucleus and their intracellular Caine Fig. H indica Parekh, , 2007 an BK Fig. B, wherem  . If the cells had been permeabilised by the bile acids, [Ca2+]i should have increased dramatically (in fact to 100 μm). Finally, Gd3+ abolished Ca2+ signal generation , which indicates that Ca2+ influx occurs via ion channels. Therefore it is unlikely that the effects of bile acids on [Ca2+]i in PSCs were caused by their detergent properties.Bile acid salts in high millimolar concentrations have detergent properties and are effective membrane permeabilising agents, often used as components of cell lysis buffers Linke, . TherefoSCs Fig. C. If th+ gradient across the plasma membrane : (1) the sodium–taurocholate cotransporting polypeptide , predominantly present in hepatocytes; (2) the apical sodium‐dependent bile acid transporter , which is expressed in the intestine; (3) and the sodium‐dependent organic anion transporter . SOAT, whose expression was found to be relatively high in the pancreas, is able to transport sulfo‐conjugated bile acids such as TLC‐S , which could indicate differences in expression of NTCP between different species.Naet al. et al. et al. et al. +‐dependent bile acid transport in the pancreas could provide a pharmacological tool useful in the acute phase of bile‐induced pancreatitis.Since NTCP serves as an entry receptor for hepatitis B virus (HBV) . The presence of many other transporters has not been tested. However, the detailed molecular characterisation of membrane transport in PSCs is outside the scope of this work.Na4A1 Fig. A. The p2+ responses to external stimuli and there is no evidence for propagation of Ca2+ signals from one cell type to another, we found that induction of Ca2+ signals in PSCs by BK makes them susceptible to TLC‐S treatment and increases cell necrosis in PSCs as well as in neighbouring PACs . An important role for BK in the development of acute pancreatitis was proposed over half a century ago . Liberation of enzymes from PACs, including kallikreins, would generate increased levels of BK in the pancreatic tissue. In turn, BK, by inducing Ca2+ signals in PSCs, would further escalate the pathological effects of bile acids in the pancreas.Our findings also shed new light on the current understanding of the mechanisms promoting pancreatic pathology, which involve not only signalling in PACs but also in PSCs. Although these two cell types have developed independent CaACs Fig. A. An imsis Fig. D. Liberet al. et al. et al. et al. et al. et al. Finally, the fact that PSCs are very sensitive targets for bile acids might have other important implications for acute biliary pancreatitis. PSCs are responsible for development of tissue fibrosis and morphological features present in chronic injury of the pancreas .2+ signals and necrosis are the hallmark of acute biliary pancreatitis. Until now the adverse effects of the bile were attributed only to actions on pancreatic acinar cells and, to a lesser extent, duct cells. Indeed, necrotic death of acinar cells is particularly threatening for the pancreas, as it is associated with the release of activated digestive enzymes leading to extensive inflammation and autodigestion of the organ. Our data demonstrate for the first time that bile acids cause pathological Ca2+ signals and necrosis in pancreatic stellate cells, largely overlooked until recently. In fact, in this study stellate cells showed higher sensitivity, than adjacent acinar cells, to Ca2+ signals and necrotic death induced by the most abundant components of the bile: sodium cholate and taurocholate. Furthermore, triggering Ca2+ signals by bradykinin or induction of necrosis by sodium cholate in stellate cells was associated with exacerbated acinar cell death induced by taurolithocholic acid 3‐sulfate. Cholate‐ and taurocholate‐induced Ca2+ signals, as well as necrosis in stellate cells, were dependent on Na+‐driven bile uptake, most likely mediated by sodium–taurocholate cotransporting polypeptide (NTCP). Therapeutic intervention by temporary inhibition of pancreatic NTCP could therefore protect stellate cells from bile‐mediated killing and, at the same time, ameliorate necrosis in adjacent acinar cells. Our results also suggest that a substantial reduction in the severity of the early stages of acute biliary pancreatitis may be achieved by inhibition of pro‐inflammatory stimuli (such as bradykinin) that trigger Ca2+ signals in stellate cells.Excessive Ca


OS of post-LD t-AML patients was significantly influenced by age, performance status, myelodysplastic syndrome prior to LD/t-AML, and treatment regimen for LD. Thus, t-AML following lymphoid malignancies treatment should be considered as very high-risk secondary AML. New treatment strategies in patients with LD/t-AML are needed urgently.Therapy-related acute myeloid leukemia (t-AML) is a heterogeneous entity most frequently related to breast cancer or lymphoproliferative diseases (LD). Population-based studies have reported an increased risk of t-AML after treatment of lymphomas. The aim of this study was to describe the characteristics and outcome of 80 consecutive cases of t-AML following treatment of LD. t-AML accounted for 2.3% of all AML cases, occurred 60 months after LD diagnosis, and were characterized by a high frequency of FAB M6 AML and poor-risk cytogenetic abnormalities. Time to t-AML diagnosis was influenced by patient age, type of LD, and treatment. Among the 48 t-AML patients treated with intensive chemotherapy, median overall survival (OS) was 7.7 months compared to 26.1 months in  MLL] rearrangements among others. The poor prognosis of t-AML was first reported in patients with multiple myeloma treated with melphalan [Therapy-related acute myeloid leukemia (t-AML) is described in the WHO classification –2 as AMLelphalan . Subsequelphalan  whereas elphalan . Individelphalan .PML and RARA genes, leading to t translocation. Moreover, mitoxantrone has been associated with secondary AML in patients treated for a nonmalignant condition [TP53 mutations in a founding clone that expands preferentially after therapy and evolves to leukemia through the accumulation of chromosomal abnormalities found typically in t-AML [Although most of the knowledge of the relationship between prior exposure to anti-cancer therapy and therapy-related AML comes from epidemiological studies, arguments for a direct impact of cytotoxic therapy in leukemogenesis exist. In therapy-related acute promyelocytic leukemia, mitoxantrone, etoposide, and doxorubicin have been shown to induce DNA cleavage at specific genomic hot spot sites in ondition . More rein t-AML .The most frequent therapy-related myeloid neoplasms are related to breast cancer and lymphoproliferative diseases (LD) –12. DataSince previous neoplasia and treatment are highly heterogeneous in the t-AML category, the aim of this study was to describe the characteristics and outcome of AML following treatment of lymphoid malignancies.In the 1997–2012 period, 80 consecutive cases of t-AML following treatment of NHL, HL, T-cell lymphoma (TCL), and chronic lymphocytic leukemia (CLL)  were diagnosed. Median age at LD diagnosis was 60 years . Thirteen patients (16%) had diffuse large B cell lymphoma (DLBCL), five had mantle cell lymphoma (MCL) (6%), two had Burkitt-like lymphoma (BL) (3%), fourteen had follicular lymphoma (FL) (18%), two had MALT/SALT lymphoma (3%), one had lymphocytic lymphoma (1%), 11 had HL (14%), eight had CLL (10%), and five had TCL (6%)  and median bone marrow blast was 46% . Cytogenetic risk was favorable for three patients (4%), intermediate for 36 patients (45%), and adverse for 29 patients (36%). MLL rearrangements, chromosome 5 and 7 abnormalities, were observed in 6, 16, and 22% of cases respectively, whereas 22% had complex karyotypes and 22% monosomal karyotypes. These poor-risk cytogenetic abnormalities were two-fold more frequent than in de novo AML (Table de novo AML (n = 829), AML secondary to myelodysplastic syndrome, Philadelphia-negative myeloproliferative neoplasm or chronic myelomonocytic leukemia (n = 292) and t-AML following treatment of solid cancers (n = 85) diagnosed in the 2000–2012 period are presented in Table LD-therapy-related AML (LD/t-AML) accounted for 2.3% of all cases of AML treated with intensive chemotherapy in the studied period. Median time from LD diagnosis to t-AML diagnosis was 60 months . Median age at t-AML diagnosis was 66 years . Characteristics of LD/t-AML are described in Table P = 0.003). Time from LD diagnosis to LD/t-AML diagnosis was shorter for TCL, lymphocytic lymphoma, DLBCL and BL , intermediate for FL, MALT/SALT, MCL and CLL , and longer for HL (95.6 months). Table P = 0.02 and 48.6 months, P = 0.009, respectively). Neither subtype of LD nor specific regimen used for LD treatment (including rituximab regimen vs. others) influenced the cytogenetic profile of LD/t-AML. However, there was a trend for an increased incidence of monosomal karyotypes in patients who received an ASCT . 53% of patients presented one or more cytopenias between end of treatment of lymphoma and diagnosis of LD/t-AML. For these patients, time to LD/t-AML was shorter . In three cases, cytopenias occurred during lymphoma treatment: one case of neutropenia and two cases of thrombocytopenia occurred after 4, 4 or 7 cycles, or R-chemotherapy, leading to treatment withdrawal or dose reduction. For these three patients, time to AML was even shorter (26.1 months). Seventeen patients (21%) developed a documented myelodysplastic syndrome (MDS) at least six months before t-AML diagnosis.Age at LD diagnosis had a major impact on time from LD diagnosis to LD/t-AML diagnosis, which was longer for patients younger than 60 years old , azacitidine (n = 12), low dose cytarabine (n = 4), 6-mercaptopurine and low dose methotrexate (n = 3), or best supportive care (n =12). Only three patients underwent allogeneic stem cell transplantation in complete remission (CR) after intensive chemotherapy. Characteristics of patients treated with intensive chemotherapy or azacitidine are summarized in Table Treatment of LD/t-AML consisted in intensive chemotherapy n = 8, azacitWhen considering patients treated with intensive chemotherapy, CR, early death, and treatment failure rates were 54%, 13%, and 33%, respectively. Among the 24 patients with intermediate-risk cytogenetics, 13 (54%) achieved CR as did 8 out of 17 patients (47%) with adverse cytogenetics. Median OS from LD/t-AML and from LD diagnosis were 7.7 and 75.6 months, respectively.P = 0.02). Also, LD with advanced stages displayed shorter OS compared to localized stages . Similarly, number of prior treatment lines for LD was associated with a shorter median OS , although this difference was not statistically significant. In univariate analysis, age greater than 60 years (P= 0.002), ECOG performance status greater than 1 (P = 0.0002), myelodysplastic syndrome prior to LD/t-AML (P = 0.09), rituximab-containing regimens (P = 0.07), and fludarabine-based regimens or chlorambucil (P = 0.11) had a negative impact on OS  and ECOG performance status 2–3  were independent prognostic factors in patients treated by intensive chemotherapy.OS from t-AML diagnosis was highly variable according to the type of LD, with AML following treatment of DLBCL, HL and TCL displaying longer OS , compared to OS of AML following treatment of MALT/SALT, CLL, MCL, BL and FL  had a median OS of 19.7 months. Median OS was 26.1 months in de novo AML (n = 663), 4.2 months in post-Philadelphia negative myeloproliferative neoplasm AML (n = 22), 9.4 months in post-myelodysplastic syndrome AML (n = 64), 8.6 months in post-chronic myelomonocytic leukemia AML (n = 19), 13.4 months in t-AML following the treatment of solid cancer (n = 50), and 14.7 months when focusing on breast cancer only (n = 30) . This result is slightly higher than the incidence reported by the Danish and Swedish registries and by the German-Austrian AMLSG group , 13, 18.MLL rearrangements, complex and monosomal karyotypes, compared to de novo AML. In particular, FAB-M6 AML was found in 11% of cases, which is unusual in large series of AML and significantly higher compared to de novo AML (3%).Despite obvious pitfalls including heterogeneity of LD subtypes and treatments, a low number of patients, and missing data due to the long period of inclusion, this retrospective study describes all consecutive cases of AML secondary to LD treatments in a recent period and contributes to define clinical characteristics and prognostic factors for this particular subtype of t-AML since most studies included various previous cancer including but not limited to LD. As expected, we found an overrepresentation of TP53 mutations selected by previous chemotherapy, predisposing to genomic instability [FLT3-ITD found in only two of 17 cases (11.8%) and NPM1 mutation in one of 15 (6.7%). A recent study has established a genetic classification of t-AML through the sequencing of 82 genes suspected to be involved in the pathogenesis of myeloid malignancies [SRSF2, SF3B1, U2AF1, ZRSR2, ASXL1, EZH2, BCOR, STAG2 or TP53 mutations since the sequencing of these genes was not performed throughout the study period. Additional studies are needed to determine the distribution of theses mutations and provide new elements for better understanding the pathogenesis of t-AML following lymphoid malignancies.We have identified distinct profiles of LD/t-AML according to the LD subtype. Indeed, longer latency and better OS were observed in HL patients, whereas TCL or aggressive NHL patients had a shorter time to t-AML and CLL patients had the lowest median overall survival. Previous treatments and subsequent genomic or microenvironment alterations, altered immune background related to the underlying LD and treatment, age at LD diagnosis  , or genetability , 21–22. tability . In our gnancies . UnfortuThe risk of AML following B-CLL treatment remains controversial since some studies did not report such an association . It has de novo AML [Intensive chemotherapy in patients with AML secondary to previous MDS, CMML, MPN, or prior cytotoxic exposure remains unsatisfactory compared to novo AML , 19. Hownovo AML –30. Azacnovo AML –33, whicIn conclusion, the prognosis of LD/t-AML is particularly poor whatever the treatment. Prevention of AML emergence by using a less leukemogenic regimen for lymphoid malignancies should be carefully weighed at LD diagnosis. New treatment strategies in patients with LD/t-AML and other secondary AML are urgently needed.Our study included 80 consecutive cases of t-AML secondary to NHL, HL, T-cell lymphoma and chronic lymphocytic leukemia diagnosed and treated in Toulouse University Hospital and Institut Paoli-Calmettes (Marseille) between 1997 and 2012. All patients were treated for their LD. AML secondary to myeloma or acute lymphoblastic leukemia were not considered in this study. Written informed consent was obtained from all patients in accordance with the Declaration of Helsinki, allowing the collection of clinical and biological data in an anonymized database. The cytogenetic classification for AML was in accordance with the Medical Research Council classification . Treatmet-test (or Mann-Whitney U test) for quantitative data. Complete response referred to the combination of complete response (CR) and complete response with incomplete blood count recovery (CRi) defined by international consensus criteria [P values were two-sided, and the significance threshold was < 0.05. Statistical analyses were performed on GraphPad Prism and STATA v13.We described patient characteristics using number and frequency for qualitative data and median, and interquartile range (IQR) for quantitative data. Differences were tested with Chi2 test  for qualitative data and with Student criteria . Early d


This feature could underlie the relative failure of anti-VEGF therapies in cancer patients. Herein, we investigated whether and how VEGF uses Ca2+ signalling to control angiogenesis in BC-derived ECFCs (BC-ECFCs). Although VEGFR-2 was normally expressed, VEGF failed to induce proliferation and in vitro tubulogenesis in BC-ECFCs. Likewise, VEGF did not trigger robust Ca2+ oscillations in these cells. Similar to normal cells, VEGF-induced intracellular Ca2+ oscillations were triggered by inositol-1,4,5-trisphosphate-dependent Ca2+ release from the endoplasmic reticulum (ER) and maintained by store-operated Ca2+ entry (SOCE). However, InsP3-dependent Ca2+ release was significantly lower in BC-ECFCs due to the down-regulation of ER Ca2+ levels, while there was no remarkable difference in the amplitude, pharmacological profile and molecular composition of SOCE. Thus, the attenuation of the pro-angiogenic Ca2+ response to VEGF was seemingly due to the reduction in ER Ca2+ concentration, which prevents VEGF from triggering robust intracellular Ca2+ oscillations. However, the pharmacological inhibition of SOCE prevented BC-ECFC proliferation and in vitro tubulogenesis. These findings demonstrate for the first time that BC-ECFCs are insensitive to VEGF, which might explain at cellular and molecular levels the failure of anti-VEGF therapies in BC patients, and hint at SOCE as a novel molecular target for this disease.Endothelial colony forming cells (ECFCs) represent a population of truly endothelial precursors that promote the angiogenic switch in solid tumors, such as breast cancer (BC). The intracellular Ca VEGF-inechanism , which iepletion . Once ac (TRPC1) , 31, 32.ed ECFCs , 33, 34]ctively) , 35, whi release . Convers release  and IH-E release , thereby release . Of noteBC-ECFCs .2+ oscillations in BC-ECFCs. We exploited a multi-disciplinary approach, comprising electron microscopy (EM), Ca2+ imaging, real-time polymerase chain reaction (qRT-PCR), Western blotting, and functional assays to demonstrate that VEGF fails to promote proliferation and in vitro tubulogenesis in BC-ECFCs due to the down-regulation of the underlying repetitive Ca2+ spikes. The suppression of the Ca2+-dependent response to VEGF involves the decrease in ER Ca2+ levels. Conversely, SOCE is still functional in these cells and can be targeted to inhibit BC-ECFC proliferation. Our data contribute to shed light at cellular and molecular level on the failure of anti-VEGF therapies and hint at SOCE as an alternative target to halt vascularization in this disease.The present investigation was endeavoured to assess whether and how VEGF stimulates pro-angiogenic CaA recent microarray analysis unveiled that the genomic profile of BC-ECFCs was dramatically altered as compared to normal cells (N-ECFCs) as indicated by the identification of 342 differentially expressed genes  in the former . In ordein vitro tubulogenesis in ECFCs in ECFCs . Under sin ECFCs , 24, 26.shown in , 23.2+ oscillations, which develop with some delay after addition of the growth factor [2+]i also in BC-ECFCs . The percentage of responding cells was 36.9±10.2% (n=477), which was significantly lower (p=0.00339) as compared to that measured in N-ECFCs . The Ca2+ response recorded in BC-ECFCs consisted in either transient Ca2+ peaks or short, asynchronous Ca2+ oscillations, which did not arise in phase among the neighbouring cells from a given microscopic field  smaller in BC-ECFCs as compared to healthy cells. Conversely, the latency of the Ca2+ response was significantly (p<0.05) longer in BC-ECFCs . Unlike cells bathed in the presence of extracellular Ca2+  longer under 0Ca2+ conditions  (1-amino]hexyl]-1H-pyrrole-2,5-dione) , suppressed VEGF-induced Ca2+ oscillations , which selectively inhibits the activity of Sarco-Endoplasmic Reticulum Ca2+-ATPase (SERCA), thereby preventing Ca2+ sequestration and emptying the endogenous Ca2+ stores [2+ levels following 30 min preincubation with CPA in the absence of extracellular Ca2+ (0Ca2+). The statistical analysis of these data has been synthesized in Figure 2+ entry in N-ECFCs [2+ oscillations by pre-incubating the cells with BTP2 -1H-pyrazo l-1-yl]phenyl)-4-methyl-1,2,3-thiadiazol e-5-carboxamide) , a pyrazole derivative which selectively inhibits SOCE in both ECFCs i represents a central hub in the sophisticated network of interconnected signalling pathways that are recruited following VEGFR2 stimulation. For instance, intracellular Ca2+ oscillations deliver the most effective signal to engage the Ca2+-dependent transcription factors, NF-κB and nuclear factor of activated T cells (NFAT), which play a key role in VEGF-induced ECFC proliferation ER through recombinant ER-targeted aequorin ER and activates SOCs, was not sufficient to enhance SOCE in the latter, as previously shown in human salivary gland cells ER, which prevents InsP3 from triggering the dynamic interaction between InsP3-dependent Ca2+ release and SOCE which reliably engages the Ca2+-dependent pro-angiogenic genetic program in N-ECFCs. The drop in ER Ca2+ levels could be due to the up-regulation of TMTC1 recently reported in BC-ECFCs i was monitored by measuring, for each ROI, the ratio of the mean fluorescence emitted at 510 nm when exciting alternatively at 340 and 380 nm (shortly termed “ratio”). An increase in [Ca2+]i causes an increase in the ratio  f(t) dt was computed for each calcium trace using Morlet function ψ(t) ≈ π-1/4 exp(–0.5 t2) exp(ist) as mother wavelet, then, after having renamed b as t and having done the substitution ν = s/(2πa), the activity index J(t) ≈ ∫ |W|2ν dν was calculated according to equations 3,4,5 and 9 of [To give a quantitative evaluation of the differences in Caented in , 39, 40.3Rs1-3, as previously described [Total RNA was extracted from N- and BC-ECFCs by using the QIAzol Lysis Reagent . Single cDNA was synthesized from RNA (1 μg) using random hexamers and M-MLV Reverse Transcriptase . Reverse transcription was always performed in the presence or absence (negative control) of the reverse transcriptase enzyme. qRT-PCR was performed in triplicate using 1 μg cDNA and specific primers (intron-spanning primers) for Stim1-2, Orai1-3, TRPC1-7, and InsPescribed , 47 . The homogenates were solubilized in LaemmLi buffer  and 30 μg proteins were separated on 10% SDS-polyacrilamide gel electrophoresis and transferred to the Hybond-P PVDF Membrane  by electroelution. After 1 h blocking with Tris buffered saline (TBS) containing 3% BSA and 0.1% Tween (blocking solution), the membranes were incubated for 3 h at room temperature with the following affinity purified antibodies diluted 1:200 in the TBS and 0.1% Tween: anti-Stim1 (sc-166840), anti-Orai1 (sc-68895), anti-TRPC1 (sc-133076), anti-TRPC3/6/7 (sc-15056), and anti-IP3R-I/II/III (sc-377518) from Santa Cruz Biotechnology, anti-Orai3 (HPA015022), anti-Stim2 (PRS4123) from Sigma-Aldrich , and anti β-actin rabbit antibody as control . The membranes were washed and incubated for 1 h with peroxidase-conjugated mouse, rabbit or goat IgG (1:120000 in blocking solution), from Dakocytomation (P0260), Chemicon (AP132P), and Santa Cruz (sc-2354), respectively. The bands were detected with the ECL™ Select western blotting detection system . Prestained molecular weight markers  were used to estimate the molecular weight of the bands. Control experiments were performed by using the antibody preadsorbed with a 20-fold molar excess of the immunizing peptide or by incubating the blots with non immune serum.Protein contents of all the samples were determined by the Bradford's method using bovine serum albumin (BSA) as standard , 47.EBM and EGM-2 were purchased from Clonetics . Fura-2/AM was obtained from Molecular Probes . BTP2 was purchased from Calbiochem . All other chemicals were obtained from Sigma Chemical Co. .5 ECFCs-derived cells (first passage) in 30-mm collagen-treated dishes in the presence of EBM-2 and 5% FBS supplemented with 10 ng/mL VEGF and one of the either compounds: BAPTA , BTP2 , La3+ , or CAI . Cultures were incubated at 37°C (in 5% CO2 and humidified atmosphere) and cell growth assessed every day until confluence was reached in control cultures. At this point, cells were recovered by trypsinization from all dishes and the cell number assessed by counting in a haemocytometer. Preliminary experiments showed no unspecific or toxic effect for each agent when used at these concentrations. Each assay was repeated in triplicate.As described elsewhere , 87, gro4 per well in Cultrex (Trevigen)-coated 96 well plates, in the presence of EBM-2 ± 10 ng/mL VEGF. Capillary network formation was assessed starting from 4 to 24 hours later. Three different sets of experiments, each performed in duplicate, were carried out. Experiments were repeated a minimum of three times and the vasculogenic response was measured by evaluating both dimensional and topological parameters. As illustrated in [in vitro angiogenesis with the EGM-2 medium in the presence and absence of the following drugs: BAPTA , BTP2 , or CAI .The tube formation assay was carried out as previously shown . To evalrated in , we analrated in . To eval