Fibroblast Growth Factor-2 Antagonist Activity and Angiostatic Capacity of Sulfated Escherichia coli K5 Polysaccharide Derivatives *

The angiogenic basic fibroblast growth factor (FGF2) interacts with tyrosine kinase receptors (FGFRs) and heparan sulfate proteoglycans (HSPGs) in endothelial cells. Here, we report the FGF2 antagonist and antiangiogenic activity of novel sulfated derivatives of the Escherichia coli K5 polysaccharide. K5 polysaccharide was chemically sulfated in N- and/or O-position after N-deacetylation. O-Sulfated and N,O-sulfated K5 derivatives with a low degree and a high degree of sulfation compete with heparin for binding to 125I-FGF2 with different potency. Accordingly, they abrogate the formation of the HSPG.FGF2.FGFR ternary complex, as evidenced by their capacity to prevent FGF2-mediated cell-cell attachment of FGFR1-overexpressing HSPG-deficient Chinese hamster ovary (CHO) cells to wild-type CHO cells. They also inhibited 125I-FGF2 binding to FGFR1-overexpressing HSPG-bearing CHO cells and adult bovine aortic endothelial cells. K5 derivatives also inhibited FGF2-mediated cell proliferation in endothelial GM 7373 cells and in human umbilical vein endothelial (HUVE) cells. In all these assays, the N-sulfated K5 derivative and unmodified K5 were poorly effective. Also, highly O-sulfated and N,O-sulfated K5 derivatives prevented the sprouting of FGF2-transfected endothelial FGF2-T-MAE cells in fibrin gel and spontaneous angiogenesis in vitro on Matrigel of FGF2-T-MAE and HUVE cells. Finally, the highly N,O-sulfated K5 derivative exerted a potent antiangiogenic activity on the chick embryo chorioallantoic membrane. These data demonstrate the possibility of generating FGF2 antagonists endowed with antiangiogenic activity by specific chemical sulfation of bacterial K5 polysaccharide. In particular, the highly N,O-sulfated K5 derivative may provide the basis for the design of novel angiostatic compounds

Phosgene distribution derived from MIPAS ESA v8 data: intercomparisons and trends

The Michelson Interferometer for Passive Atmospheric Sounding (MIPAS) measured the middle-infrared limb emission spectrum of the atmosphere from 2002 to 2012 on board ENVISAT, a polar-orbiting satellite. Recently, the European Space Agency (ESA) completed the final reprocessing of MIPAS measurements, using version 8 of the level 1 and level 2 processors, which include more accurate models, processing strategies, and auxiliary data. The list of retrieved gases has been extended, and it now includes a number of new species with weak emission features in the MIPAS spectral range. The new retrieved trace species include carbonyl chloride (COCl2), also called phosgene. Due to its toxicity, its use has been reduced over the years; however, it is still used by chemical industries for several applications. Besides its direct injection in the troposphere, stratospheric phosgene is mainly produced from the photolysis of CCl4, a molecule present in the atmosphere because of human activity. Since phosgene has a long stratospheric lifetime, it must be carefully monitored as it is involved in the ozone destruction cycles, especially over the winter polar regions. In this paper we exploit the ESA MIPAS version 8 data in order to discuss the phosgene distribution, variability, and trends in the middle and lower stratosphere and in the upper troposphere. The zonal averages show that phosgene volume mixing ratio is larger in the stratosphere, with a peak of 40 pptv (parts per trillion by volume) between 50 and 30 hPa at equatorial latitudes, while at middle and polar latitudes it varies from 10 to 25 pptv. A moderate seasonal variability is observed in polar regions, mostly between 80 and 50 hPa. The comparison of MIPAS–ENVISAT COCl2 v8 profiles with the ones retrieved from MIPAS balloon and ACE-FTS (Atmospheric Chemistry Experiment – Fourier Transform Spectrometer) measurements highlights a negative bias of about 2 pptv, mainly in polar and mid-latitude regions. Part of this bias is attributed to the fact that the ESA level 2 v8 processor uses an updated spectroscopic database. For the trend computation, a fixed pressure grid is used to interpolate the phosgene profiles, and, for each pressure level, VMR (volume mixing ratio) monthly averages are computed in pre-defined 10∘ wide latitude bins. Then, for each latitudinal bin and pressure level, a regression model has been fitted to the resulting time series in order to derive the atmospheric trends. We find that the phosgene trends are different in the two hemispheres. The analysis shows that the stratosphere of the Northern Hemisphere is characterized by a negative trend of about −7 pptv per decade, while in the Southern Hemisphere phosgene mixing ratios increase with a rate of the order of +4 pptv per decade. This behavior resembles the stratospheric trend of CCl4, which is the main stratospheric source of COCl2. In the upper troposphere a positive trend is found in both hemispheres.</p

Monomeric gremlin is a novel vascular endothelial growth factor receptor-2 antagonist

Angiogenesis plays a key role in various physiological and pathological conditions, including inflammation and tumor growth. The bone morphogenetic protein (BMP) antagonist gremlin has been identified as a novel pro-angiogenic factor. Gremlin promotes neovascular responses via a BMP-independent activation of the vascular endothelial growth factor (VEGF) receptor-2 (VEGFR2). BMP antagonists may act as covalent or non-covalent homodimers or in a monomeric form, while VEGFRs ligands are usually dimeric. However, the oligomeric state of gremlin and its role in modulating the biological activity of the protein remain to be elucidated.Here we show that gremlin is expressed in vitro and in vivo both as a monomer and as a covalently linked homodimer. Mutagenesis of amino acid residue Cys141 prevents gremlin dimerization leading to the formation of gremlinC141A monomers. GremlinC141A monomer retains a BMP antagonist activity similar to the wild-type dimer, but is devoid of a significant angiogenic capacity. Notably, we found that gremlinC141A mutant engages VEGFR2 in a non-productive manner, thus acting as receptor antagonist. Accordingly, both gremlinC141A and wild-type monomers inhibit angiogenesis driven by dimeric gremlin or VEGF-A165. Moreover, by acting as a VEGFR2 antagonist, gremlinC141A inhibits the angiogenic and tumorigenic potential of murine breast and prostate cancer cells in vivo.In conclusion, our data show that gremlin exists in multiple forms endowed with specific bioactivities and provide new insights into the molecular bases of gremlin dimerization. Furthermore, we propose gremlin monomer as a new inhibitor of VEGFR2 signalling during tumor growth

The ESA MIPAS/Envisat level2-v8 dataset: 10 years of measurements retrieved with ORM v8.22

The observations acquired during the full mission of the Michelson Interferometer for Passive Atmospheric Sounding (MIPAS) instrument, aboard the European Space Agency Environmental Satellite (Envisat), have been analysed with version 8.22 of the Optimised Retrieval Model (ORM), originally developed as the scientific prototype of the ESA level-2 processor for MIPAS observations. The results of the analyses have been included into the MIPAS level-2 version 8 (level2-v8) database containing atmospheric fields of pressure, temperature, and volume mixing ratio (VMR) of MIPAS main targets H$_{2}$O, O$_{3}$, HNO$_{3}$, CH$_{4}$, N$_{2}$O, and NO$_{2}$, along with the minor gases CFC-11, ClONO$_{2}$, N$_{2}$O$_{5}$, CFC-12, COF$_{2}$, CCl$_{4}$, CF$_{4}$, HCFC-22, C$_{2}$H$_{2}$, CH$_{3}$Cl, COCl$_{2}$, C$_{2}$H$_{6}$, OCS, and HDO. The database covers all the measurements acquired by MIPAS in the nominal measurement mode of the full resolution (FR) part of the mission (from July 2002 to March 2004) and all the observation modes of the optimised resolution (OR) part (from January 2005 to April 2012). The number of species included in the MIPAS level2-v8 dataset makes it of particular importance for the studies of stratospheric chemistry. The database is considered by ESA the final release of the MIPAS level-2 products. The ORM algorithm is operated at the vertical grid coincident to the tangent altitudes of the observations or to a subset of them, spanning (in the nominal mode) the altitude range from 6 to 68 km in the FR phase and from 6 to 70 km in the OR period. In the latitude domain, FR profiles are spaced by about 4.7∘, while the OR profiles are spaced by about 3.7∘. For each retrieved species, the auxiliary data and the retrieval choices are described. Each product is characterised in terms of the retrieval error, spatial resolution, and “useful” vertical range in both phases of the MIPAS mission. These depend on the characteristics of the measurements (spectral and vertical resolution of the measurements), the retrieval choices (number of spectral points included in the analyses, number of altitudes included in the vertical retrieval grid), and the information content of the measurements for each trace species. For temperature, water vapour, ozone, and nitric acid, the number of degrees of freedom is significantly larger in the OR phase than in the FR one, mainly due to the finer vertical measurement grid. In the FR phase, some trace species are characterised by a smaller retrieval error with respect to the OR phase, mainly due to the larger number of spectral points used in the analyses, along with the reduced vertical resolution. The way of handling possible caveats (negative VMR, vertical grid representation) is discussed. The quality of the retrieved profiles is assessed through four criteria, two providing information on the successful convergence of the retrieval iterations, one on the capability of the retrieval to reproduce the measurements, and one on the presence of outliers. An easy way to identify and filter the problematic profiles with the information contained in the output files is provided. MIPAS level2-v8 data are available to the scientific community through the ESA portal (https://doi.org/10.5270/EN1-c8hgqx4)

Level 2 processor and auxiliary data for ESA Version 8 final full mission analysis of MIPAS measurements on ENVISAT

High quality long-term data sets of altitude-resolved measurements of the atmospheric composition are important because they can be used both to study the evolution of the atmosphere and as a benchmark for future missions. For the final ESA reprocessing of MIPAS (Michelson Interferometer for Passive Atmospheric Sounding) on ENVISAT (ENViromental SATellite) data, numerous improvements were implemented in the Level 2 (L2) processor Optimised Retrieval Model (ORM) version 8.22 (V8) and its auxiliary data. The implemented changes involve all aspects of the processing chain, from the modelling of the measurements with the handling of the horizontal inhomogeneities along the line of sight to the use of the optimal estimation technique to retrieve the minor species, from a more sensitive approach to detecting the spectra affected by clouds to a refined method for identifying low quality products. Improvements in the modelling of the measurements were also obtained with an update of the used spectroscopic data and of the databases providing the a priori knowledge of the atmosphere. The HITRAN_mipas_pf4.45 spectroscopic database was finalised with new spectroscopic data verified with MIPAS measurements themselves, while recently measured cross-sections were used for the heavy molecules. The Level 2 Initial Guess (IG2) data set, containing the climatology used by the MIPAS L2 processor to generate the initial guess and interfering species profiles when the retrieved profiles from previous scans are not available, was improved taking into account the diurnal variation of the profiles defined using climatologies from both measurements and models. Horizontal gradients were generated using the ECMWF ERA-Interim data closest in time and space to the MIPAS data. Further improvements in the L2 V8 products derived from the use of the L1b V8 products, which were upgraded to reduce the instrumental temporal drift and to handle the abrupt changes in the calibration gain. The improvements introduced into the ORM V8 L2 processor and its upgraded auxiliary data, together with the use of the L1b V8 products, lead to the generation of the MIPAS L2 V8 products, which are characterised by an increased accuracy, better temporal stability and a greater number of retrieved species

Level 2 processor and auxiliary data for ESA Version 8 final full mission analysis of MIPAS measurements on ENVISAT

High quality long-term data sets of altitude-resolved measurements of the atmospheric composition are important because they can be used both to study the evolution of the atmosphere and as a benchmark for future missions. For the final ESA reprocessing of MIPAS (Michelson Interferometer for Passive Atmospheric Sounding) on ENVISAT (ENViromental SATellite) data, numerous improvements were implemented in the Level 2 (L2) processor Optimised Retrieval Model (ORM) version 8.22 (V8) and its auxiliary data. The implemented changes involve all aspects of the processing chain, from the modelling of the measurements with the handling of the horizontal inhomogeneities along the line of sight to the use of the optimal estimation technique to retrieve the minor species, from a more sensitive approach to detecting the spectra affected by clouds to a refined method for identifying low quality products. Improvements in the modelling of the measurements were also obtained with an update of the used spectroscopic data and of the databases providing the a priori knowledge of the atmosphere. The HITRAN_mipas_pf4.45 spectroscopic database was finalised with new spectroscopic data verified with MIPAS measurements themselves, while recently measured cross-sections were used for the heavy molecules. The Level 2 Initial Guess (IG2) data set, containing the climatology used by the MIPAS L2 processor to generate the initial guess and interfering species profiles when the retrieved profiles from previous scans are not available, was improved taking into account the diurnal variation of the profiles defined using climatologies from both measurements and models. Horizontal gradients were generated using the ECMWF ERA-Interim data closest in time and space to the MIPAS data. Further improvements in the L2 V8 products derived from the use of the L1b V8 products, which were upgraded to reduce the instrumental temporal drift and to handle the abrupt changes in the calibration gain. The improvements introduced into the ORM V8 L2 processor and its upgraded auxiliary data, together with the use of the L1b V8 products, lead to the generation of the MIPAS L2 V8 products, which are characterised by an increased accuracy, better temporal stability and a greater number of retrieved species

Biochemical bases of the interaction of the human basic fibroblast growth factor with glycosaminoglycans: new insights from trypsin digestion studies

Heparins from bovine mucosa and lung, and chemically modified heparins were assayed for their capacity to: (i) protect human recombinant basic fibroblast growth factor (bFGF) from tryptic cleavage; (ii) prevent 1251-bFGF binding to heparan sulphate proteoglycans present in the extracellular matrix and on the cell surface of fetal bovine aortic endothelial GM 7373 cell cultures; (iii) affect 1251-bFGF binding to high-affinity tyrosine kinase FGF receptors present on the cell membrane of GM 7373 cells; (iv) inhibit the mitogenic activity exerted by bFGF in the same cells. The results demonstrate thatthe potency shown by mucosal heparins in the different assays is a direct function of size, verylow- molecular-mass heparin (2.0 kDa) being significantly less effective on a molar basis than unfractionated heparin (13.6 kDa). Increased flexibility of the backbone structure, as observed in reduced/oxidized heparins of different size, does not affect the capacity of the polysaccharide to interact with bFGF. In contrast, selective 2-O-desulphation, but not 6-O-desulphation, drastically reduced the capacity of heparin to protect bFGF from proteolytic cleavage, to affect its interaction with low- and high-affinity sites, and to inhibit its mitogenic activity. Two preparations of bovine lung heparin, differing in molecular mass, were as effective as mucosal heparin in the bFGF-tryptic-digestion assay and the endothelial-cell proteoglycan-binding assay, but they were highly inefficient at inhibiting the capacity of bFGF to interact with its tyrosine kinase receptors. Bovine lung heparins were also less effective than mucosal heparin as bFGF antagonists in GM 7373-cell-proliferation assays. N-Desulphated/N-acetylated bovine lung heparin retained only a significant capacity to protect bFGF from tryptic cleavage. The results demonstrate that different chemical features of the heparin molecule, including decrease in molecular mass, selective desulphation, disaccharide composition and clustering, affect differently the capacity of the glycosaminoglycan to interact with bFGF and to influence its biological behaviour in different assays in vitro and in endothelial cell cultures. Our findings should aid the design of synthetic oligosaccharides aimed at improving the. bioavailability of bFGF when administered in vivo as a therapeutic agent

Fibroblast growth factor-2 antagonist activity and angiostatic capacity of sulfated Escherichia coli K5 polysaccharide derivatives.

The angiogenic basic fibroblast growth factor (FGF2) interacts with tyrosine kinase receptors (FGFRs) and heparan sulfate proteoglycans (HSPGs) in endothelial cells. Here, we report the FGF2 antagonist and antiangiogenic activity of novel sulfated derivatives of the Escherichia coli K5 polysaccharide. K5 polysaccharide was chemically sulfated in N- and/or O-position after N-deacetylation. O-Sulfated and N,O-sulfated K5 derivatives with a low degree and a high degree of sulfation compete with heparin for binding to I-125-FGF2 with different potency. Accordingly, they abrogate the formation of the HSPG.FGF2.FGFR ternary complex, as evidenced by their capacity to prevent FGF2-mediated cell-cell attachment of FGFR1-overexpressing HSPG-deficient Chinese hamster ovary (CHO) cells to wildtype CHO cells. They also inhibited I-125-FGF2 binding to FGFR1-overexpressing HSPG-bearing CHO cells and adult bovine aortic endothelial cells. K5 derivatives also inhibited FGF2-mediated cell proliferation in endothelial GM 7373 cells and in human umbilical vein endothelial (HUVE) cells. In all these assays, the N-sulfated K5 derivative and unmodified K5 were poorly effective. Also, highly O-sulfated and N,O-sulfated K5 derivatives prevented the sprouting of FGF2-transfected endothelial FGF2-T-MAE cells in fibrin gel and spontaneous angiogenesis in vitro on Matrigel of FGF2-T-MAE and HUVE cells. Finally, the highly N,O-sulfated K5 derivative exerted a potent antiangiogenic activity on the chick embryo chorioallantoic membrane. These data demonstrate the possibility of generating FGF2 antagonists endowed with antiangiogenic activity by specific chemical sulfation of bacterial K5 polysaccharide. In particular, the highly N,O-sulfated K5 derivative may provide the basis for the design of novel angiostatic compounds