M-CSF Signals through the MAPK/ERK Pathway via Sp1 to Induce VEGF Production and Induces Angiogenesis In Vivo

BACKGROUND: M-CSF recruits mononuclear phagocytes which regulate processes such as angiogenesis and metastases in tumors. VEGF is a potent activator of angiogenesis as it promotes endothelial cell proliferation and new blood vessel formation. Previously, we reported that in vitro M-CSF induces the expression of biologically-active VEGF from human monocytes. METHODOLOGY AND RESULTS: In this study, we demonstrate the molecular mechanism of M-CSF-induced VEGF production. Using a construct containing the VEGF promoter linked to a luciferase reporter, we found that a mutation reducing HIF binding to the VEGF promoter had no significant effect on luciferase production induced by M-CSF stimulation. Further analysis revealed that M-CSF induced VEGF through the MAPK/ERK signaling pathway via the transcription factor, Sp1. Thus, inhibition of either ERK or Sp1 suppressed M-CSF-induced VEGF at the mRNA and protein level. M-CSF also induced the nuclear localization of Sp1, which was blocked by ERK inhibition. Finally, mutating the Sp1 binding sites within the VEGF promoter or inhibiting ERK decreased VEGF promoter activity in M-CSF-treated human monocytes. To evaluate the biological significance of M-CSF induced VEGF production, we used an in vivo angiogenesis model to illustrate the ability of M-CSF to recruit mononuclear phagocytes, increase VEGF levels, and enhance angiogenesis. Importantly, the addition of a neutralizing VEGF antibody abolished M-CSF-induced blood vessel formation. CONCLUSION: These data delineate an ERK- and Sp1-dependent mechanism of M-CSF induced VEGF production and demonstrate for the first time the ability of M-CSF to induce angiogenesis via VEGF in vivo

Visualization of Murine Intranasal Dosing Efficiency Using Luminescent Francisella tularensis: Effect of Instillation Volume and Form of Anesthesia

Intranasal instillation is a widely used procedure for pneumonic delivery of drugs, vaccine candidates, or infectious agents into the respiratory tract of research mice. However, there is a paucity of published literature describing the efficiency of this delivery technique. In this report we have used the murine model of tularemia, with Francisella tularensis live vaccine strain (FTLVS) infection, to evaluate the efficiency of pneumonic delivery via intranasal dosing performed either with differing instillation volumes or different types of anesthesia. FTLVS was rendered luminescent via transformation with a reporter plasmid that constitutively expressed the Photorhabdus luminescens lux operon from a Francisella promoter. We then used an IVIS Spectrum whole animal imaging system to visualize FT dissemination at various time points following intranasal instillation. We found that instillation of FT in a dose volume of 10 µl routinely resulted in infection of the upper airways but failed to initiate infection of the pulmonary compartment. Efficient delivery of FT into the lungs via intranasal instillation required a dose volume of 50 µl or more. These studies also demonstrated that intranasal instillation was significantly more efficient for pneumonic delivery of FTLVS in mice that had been anesthetized with inhaled (isoflurane) vs. parenteral (ketamine/xylazine) anesthesia. The collective results underscore the need for researchers to consider both the dose volume and the anesthesia type when either performing pneumonic delivery via intranasal instillation, or when comparing studies that employed this technique

The development and validation of a scoring tool to predict the operative duration of elective laparoscopic cholecystectomy

Background: The ability to accurately predict operative duration has the potential to optimise theatre efficiency and utilisation, thus reducing costs and increasing staff and patient satisfaction. With laparoscopic cholecystectomy being one of the most commonly performed procedures worldwide, a tool to predict operative duration could be extremely beneficial to healthcare organisations. Methods: Data collected from the CholeS study on patients undergoing cholecystectomy in UK and Irish hospitals between 04/2014 and 05/2014 were used to study operative duration. A multivariable binary logistic regression model was produced in order to identify significant independent predictors of long (> 90 min) operations. The resulting model was converted to a risk score, which was subsequently validated on second cohort of patients using ROC curves. Results: After exclusions, data were available for 7227 patients in the derivation (CholeS) cohort. The median operative duration was 60 min (interquartile range 45–85), with 17.7% of operations lasting longer than 90 min. Ten factors were found to be significant independent predictors of operative durations > 90 min, including ASA, age, previous surgical admissions, BMI, gallbladder wall thickness and CBD diameter. A risk score was then produced from these factors, and applied to a cohort of 2405 patients from a tertiary centre for external validation. This returned an area under the ROC curve of 0.708 (SE = 0.013, p  90 min increasing more than eightfold from 5.1 to 41.8% in the extremes of the score. Conclusion: The scoring tool produced in this study was found to be significantly predictive of long operative durations on validation in an external cohort. As such, the tool may have the potential to enable organisations to better organise theatre lists and deliver greater efficiencies in care

An examination of the mixing of low-lying excited 0+ states in 116sn

The even-even tin isotopes are known to exhibit shape coexistence, the phenomenon where multiple shapes coexist in a narrow energy region at relatively low-lying levels of the nucleus. These nuclei have a 0+ spherical ground state and multipleexcited 0+ states, one of which is a band head for a deformed rotational band, caused by the promotion of two protons across the Z=50 shell gap. Experimental and theoretical investigations have been performed on 116Sn to describe the nature of the mixing that occurs between the vibrational phonon levels and the deformed rotational band by probing the character of the excited 0+ states. At the time it was thought that the 0+ states showed almost equal mixing of rotational and vibrational character, but this result was based on an indirect observation and fit of the intensity of a weak 85 keV transition. The current work, a high-statistics 116Sn measurement, demonstrates unequal mixing of character between the two excited 0+ states based on a direct measurement of the intensity of the 85 keV transition. These new results might prompt a new interpretation of the structure of 116Sn. The experiment to investigate the low-lying structure of 116Sn was conducted at TRIUMF, Canada’s National Laboratory for Nuclear and Particle Physics. A high-intensity and high-purity beam of 116In was used to populate states in 116Sn via beta decay. The resulting gamma rays were observed with the 8π detector array, which consists of twenty high-purity Compton-suppressed germanium detectors coupled to a suite of ancillary detectors for β particle detection and conversion electron spectroscopy. From this high-statistics measurement 57 gamma-ray transitions were observed,with 4 new transitions that depopulate the 3096 keV level observed for the first time with energies of 101 keV, 296 keV, 447 keV, and 871 keV. Branching ratios were determined for all of the observed transitions. For the 57 transitions observed, a relative intensity had not been reported for 17 of them, and a branching ratio had not been reported for 12 of them. Transition rates were determined for 25 transitions that depopulate levels with previously reported lifetimes, and 2 of these transition rates had not been previously observed

The investigation of the beta decay of 46K: Detailed spectroscopy of the low-lying structure of 46Ca with the GRIFFIN Spectrometer

The calcium region is currently a new frontier for modern shell model calculations, and detailed experimental data from these nuclei is critical for a comprehensive understanding of the region.Due to its very low natural abundance of 0.004%, the structure of the magic nucleus 46Ca has not been studied in great detail. Some excited states were previously identified by various reaction mechanisms, and few gamma rays were placed in the level scheme from results of beta-decay experiments equipped with limited detection capabilities. A high-statistics data set of the beta decay of the 46K 2- ground state into the excited states of 46Ca was measured with the GRIFFIN spectrometer located at TRIUMF-ISAC in December of 2014. A radioactive beam consisting almost entirely of 46K was implanted at the center of the GRIFFIN array, and the emitted gamma rays were detected by 15 high-purity germanium clover detectors. From forty hours of data collection, 430 million gamma-gamma coincidences were observed and analysed to construct the 46Ca level scheme. In total, 194 gamma rays were identified and placed into the level scheme; 150 of these transitions were observed for the first time. Angular correlations between pairs of gamma rays were analysed to investigate the spin assignments of the observed excited states. Correlations were investigated for 18 of the 42 observed excited states, and it was possible to confirm 7 previously reported spin assignments, and assign 3 new spins of 3-, 2-, and 3- for the 4435, 5052, and 5535 keV states, respectively. The measured half-life of the 96.41(10) s is in agreement with previous results. From the observed beta feeding intensities of this work, it is suggested that the 46K 2- ground state may contain more proton s1/2 character than has been previously believed. This is due to the strong population of the 5052 keV 2$^-$ state and the absence of observed feeding to the 46Ca ground state

Assessment of the Second-Ionization Potential of Lawrencium: Investigating the End of the Actinide Series with a One-Atom-at-a-Time Gas-Phase Ion Chemistry Technique.

Experiments were performed at the Lawrence Berkeley National Laboratory 88-Inch Cyclotron facility to investigate the electron-transfer reduction reaction of dipositive Lr (Z = 103) with O2 gas. Ions of 255Lr were produced in the fusion-evaporation reaction 209Bi(48Ca,2n) 255Lr and were studied with a novel gas-phase ion chemistry technique. The produced 255Lr2+ ions were trapped and O2 gas was introduced, such that the charge-exchange reaction to reduce 255Lr2+ to 255Lr1+ was observed and the reaction rate constant was determined to be k = 1.5(7) × 10-10 cm3/mol/s. The observation that this reaction proceeds establishes the lower limit on the second ionization potential of Lr to be 13.3(3) eV. This gives further support that the actinide series terminates with Lr. Additionally, this result can be used to better interpret the situation concerning the placement of Lu and Lr on the periodic table within the current framework of the actinide hypothesis. The success of this experimental approach now identifies unique opportunities for future gas-phase reaction studies on actinide and super heavy elements

M-CSF induced VEGF production occurs through a HIF-independent mechanism.

<p>A) The 1.5 kb wild type VEGF promoter <i>(WT1.5 kb)</i> and the same promoter sequence containing a non-functional mutation of the hypoxia regulatory element (HRE) <i>(ΔHRE)</i> were inserted into the pGL3-basic vector to create constructs that produce luciferase upon VEGF promoter activation. Nucleotides changed from the original wild type sequence are designated in bold. Human monocytes were transfected with either the control empty construct (pGL3-Basic), or pGL3 containing 1 µg of each of the described VEGF promoter constructs above. B) Transfected monocytes containing <i>(WT1.5 kb)</i> or <i>(ΔHRE)</i> were allowed to adhere for 1 hour in RPMI/5% FBS followed by the addition of fresh media containing rhM-CSF (100 ng/ml) for 16 hours. The adherent cells were lysed and assayed for luciferase production using a luminometer. This data represents the mean+/−SEM of six individual blood donors. All data is represented as the fold change in luciferase over the pGL3 control construct.</p

M-CSF induces Sp1 nuclear localization in an ERK-dependent manner.

<p>A) Human monocytes were left non-stimulated <i>(NS)</i> or stimulated with rhM-CSF (100 ng/ml) <i>(M-CSF)</i> for 30 minutes. Nuclear lysates were isolated and normalized for total protein. Sp1 that translocated into the nucleus in response to M-CSF was analyzed using a biotinylated Sp1 DNA sequence bound to a streptavidin-coated plate, a polyclonal rabbit anti-Sp1 primary antibody, a HRP-conjugated goat anti-rabbit IgG secondary antibody, and TMB substrate. The absorbance was read at 450 nm to reflect Sp1 within the nucleus. These data represent the mean±SEM from four independent blood donors. B) Human monocytes were starved for 6 hours, inhibited for 30 minutes with U0126 (10 µM) or DMSO (vehicle control) and left non-stimulated <i>(Non-stimulated)</i> or treated with rhM-CSF for 6 hours <i>(M-CSF+DMSO)</i> and <i>(M-CSF+U0126)</i>. The cells were fixed, permeablized, and stained with a normal IgG control antibody <i>(top row)</i> or a primary antibody targeting Sp1 followed by subsequent staining with Alexa 594-conjugated secondary antibody, targeting Sp1 <i>(red)</i>, and with DAPI stain designating the nucleus <i>(blue)</i>. Images were captured using the Zeiss LSM 510 confocal microscope. These pictures are representative of four individual monocyte donors. C) Quantification of Sp1 localization to the nucleus of monocytes (horseshoe-shaped nuclei) using Image J software. This data represents mean+/−SEM of cells from four individual trials.</p

M-CSF-induced VEGF production from human monocytes is blocked by ERK inhibition and mithramycin.

<p>A) Monocytes were pre-incubated with DMSO (vehicle control), 1 µM or 10 µM the PI3 kinase inhibitor (LY294002) for 30 minutes. The monocytes were either left non-stimulated <i>(NS)</i>, stimulated with rhM-CSF (100 ng/ml) <i>(M-CSF)</i>, with rhM-CSF (100 ng/ml) plus DMSO <i>(M-CSF+DMSO)</i>, or rhM-CSF (100 ng/ml) plus 1 or 10 µM LY294002 <i>(1)</i> and <i>(10)</i> for 48 hours. Cell-free supernatants were evaluated for VEGF by ELISA. These data represent the mean±SEM from four independent donors. B) Monocytes were pre-incubated with DMSO (vehicle control) or 10 µM of the inhibitor of ERK activity (U0126) for 30 minutes. The monocytes were either left non-stimulated <i>(NS)</i>, stimulated with rhM-CSF (100 ng/ml) plus DMSO <i>(M-CSF+DMSO)</i> or with rhM-CSF (100 ng/ml) plus U0126 <i>(M-CSF+U0126 (10 µM))</i> for 48 hours. Cell-free supernatants were evaluated for VEGF by ELISA. These data represent the mean±SEM from six independent donors. C) Freshly-isolated human monocytes were plated overnight in 5% FBS and M-CSF (20 ng/ml). The next day the cells were starved for 2 hours in minimal media. For the last 30 minutes, DMSO (vehicle control) or 10 µM U0126 was added to the appropriate samples. The cells were left non-stimulated <i>(NS)</i>, stimulated with rhM-CSF (100 ng/ml) <i>(M-CSF+DMSO)</i> or <i>(M-CSF+U0126)</i> for 10 minutes. Cell lysates were probed for phospho-ERK MAP Kinase (Thr202) and total ERK MAP Kinase by western blot analysis. This data is representative of two independent monocyte donors. D) Monocytes were pre-incubated with methanol (vehicle control) or 8, 40, or 200 nM of the Sp1 transcription factor binding inhibitor, mithramycin, for 45 minutes. The cells were either left non-stimulated <i>(NS)</i> or stimulated with rhM-CSF (100 ng/ml) <i>(M-CSF)</i>, <i>(M-CSF+MeOH)</i>, <i>(M-CSF plus 8 nM, 40 nM, or 200 nM mithramycin)</i> for 48 hours. Cell-free supernatants were evaluated for VEGF by ELISA. These data represent the mean±SEM from ten independent blood donors.</p