Human H-kininogen (120 kDa) plays a role in many pathophysiological processes and interacts with the cell surface through protein receptors and proteoglycans, which mediate H-kininogen endocytosis. In the present work we demonstrate that H-kininogen containing bradykinin domain is internalized and different endogenous kininogenases are present in CHO-K1 cells. We used CHO-K1 (wild type) and CHO-745 (mutant deficient in proteoglycans biosynthesis) cell lines. H-kininogen endocytosis was studied using confocal microscopy, and its hydrolysis by cell lysate fraction was determined by immunoblotting. Bradykinin release was also measured by radioimmunoassay. H-kininogen interaction with the cell surface of CHO-745 cells resulted in bradykinin release by serine proteases. In CHO-K1 cells, which produce heparan and chondroitin sulfate proteoglycans, internalization of H-kininogen through its bradykinin domain can occur on lipid raft domains/caveolae. Nevertheless bradykinin-free H-kininogen was not internalized by CHO-K1 cells. The H-kininogen present in acidic endosomal vesicles in CHO-K1 was approximately 10-fold higher than the levels in CHO-745. CHO-K1 lysate fractions were assayed at pH 5.5 and intact H-kininogen was totally hydrolyzed into a 62 kDa fragment. By contrast, at an assay pH 7.4, the remained fragments were 115 kDa, 83 kDa, 62 kDa and 48 kDa in size. The antipain-Sepharose chromatography separated endogenous kininogenases from CHO-K1 lysate fraction. No difference was detected in the assays at pH 5.5 or 7.4, but the proteins in the fraction bound to the resin released bradykinin from H-kininogen. However, the proteins in the unbound fraction cleaved intact H-kininogen at other sites but did not release bradykinin. H-kininogen can interact with extravascular cells, and is internalized dependent on its bradykinin domain and cell surface proteoglycans. After internalization, H-kininogen is proteolytically processed by intracellular kininogenases. The present data also demonstrates that serine or cysteine proteases in lipid raft domains/caveolae on the CHO cell can hydrolyze H-kininogen, thus releasing kinins. Kinins, including bradykinin, kallidin (Lys-bradykinin) and Met-Lys-bradykinin, are a family of vasoactive and proinflammatory peptides. Kallidin and Met-Lys-bradykinin are converted very rapidly to bradykinin by aminopeptidases . BradykiThe precursors of kinins in mammals are high (H) and low (L) molecular weight kininogens, glycoproteins synthesized in liver that exist predominantly in blood plasma but are also found in other body fluids and organs, including the kidney, and in cells such as neutrophils. Kininogens are multifunctional and multidomain glycoproteins related to cystatins . Both native forms possess an identical N-terminal heavy chain, which corresponds to domains D1-D2-D3, a short domain D4 (the bradykinin moiety), and because of an alternative splicing of the gene transcript, two different C-terminal light chains; L-kininogen possesses a single D5 domain (D5L), whereas H-kininogen possesses a D5 domain (D5H) as well as a D6 domain. Indeed, both kininogens are encoded by a single gene  located on chromosome 3 that consists of eleven exons and ten introns . Th. Th8]. Tn injury .In vivo, the decapeptide Lys-bradykinin can be easily converted into the nonapeptide bradykinin by cleavage at the Lys–Arg bond , a, a14], aThe present study examines the interaction of H-kininogen with cells using tumor epithelial cell lineages Chinese Hamster Ovary, CHO-K1 and CHO-745, which differ from each other with respect to GAG biosynthesis . On the o-phe), goat IgG anti-mouse IgG peroxidase-conjugated and goat IgG anti-rabbit IgG peroxidase-conjugated antibodies were purchased from Sigma-Aldrich Co. . The inhibitor JA-2 [N-[1-carboxy-3-phenylpropyl]-Ala-Aib-Tyr-p-aminobenzoate], originally from Ian Smith of the Baker Heart Research Institute, Australia, was kindly provided by Prof. A.C.M. Camargo . H-kininogen was obtained from EMD Biosciences Inc. ; bradykinin-free H-kininogen was purchased from Enzyme Research Laboratories . Lyso Tracker (LT) Red DND-99, 4’-6-diamidino-2-phenylindole dihydrochloride (DAPI), goat IgG anti-mouse FITC-conjugated antibody, and the Alexa Fluor 488 Protein Labeling kit were obtained from Molecular Probes/Invitrogen Detection Technologies . Molecular weight standards for SDS-electrophoresis Prestained Broad Range or Kaleidoscope Prestained molecular weight standards were purchased from Bio-Rad Laboratories Ltd. . Membrane Immobilon-P transfer membrane (PVDF) was obtained from Millipore Corporation . Fluoromont-G was purchased from Electron Microscopy Sciences . FITC-conjugated streptavidin, FITC-conjugated donkey IgG anti-rabbit IgG and Texas Red dye-conjugated streptavidin were obtained from Jackson ImmunoResearch Laboratories, Inc. . Fluoromount-G was purchased from Electron Microscopy Sciences . The Densitometer Quick Scan Flur Vis was from Helena Laboratories . Both Sephadex G-25 (PD-10 column) and AH Sepharose 4B resins were obtained from GE Healthcare .All chemicals obtained from commercial sources were of the best grade available. SuperSignal West Pico Chemiluminescent Substrate, the biotinylation kit (NHS-LC-Biotin) and EDC (1-etil–3–(3–dimetilaminopropil)carbodiimidacloridrato were purchased from Pierce Biotechnology Inc. . The inhibitors captopril, aprotinin, thiorphan, trans-epoxysuccinyl-l-leucylamido-(4-guanidino)butane (E-64), benzamidine, phenylmethylsulfonylfluoride (PMSF), antipain, 1,10-phenanthroline (363-R371) and purchased from Alexis Biochemicals ; the mouse IgG anti-kininogenD6  was kindly provided by Prof. Dr. Werner Müller-Esterl   and grown to confluence on 60-mm dishes.The epithelial cell lines CHO-K1 and CHO-745 (the mutant deficient in xylosyltransferase)  were gif3, 0.36 mM NaH2PO4, 14.7 mM HEPES, 50.0 μM Zn2+, 1.0 mM Mg2+, 2.0 mM Ca2+, 3.5 mg/mL dextrose, 3.5 mg/mL bovine serum albumin (BSA)] or (2) MES-Tyrode pH 5.5 . HK (200 nM) was incubated with cells in the absence (control) or presence of kininase inhibitors , kininase inhibitors and serine protease inhibitors  or kininase inhibitors and a cysteine protease inhibitor (9 μM E-64). After 3 h at 37°C, the incubation buffer of each sample was collected and mixed with ethanol  for 10 min at 70°C to extract kinin. Solutions were freeze-dried and dissolved in 250 μL egg albumin buffer . Aliquots (50 μL) were incubated with 100 μL of rabbit IgG anti-bradykinin  and [125I]-labeled Tyr-bradykinin (100 μL) for 20 h at 4°C. In total, 400 μL of 0.1% bovine γ-globulin in 0.01 M phosphate buffer pH 7.0, 0.14 M NaCl, and 0.1% NaN3 and 800 μL of 25% polyethylene glycol 6000 solution were added to the samples, which were incubated for 10 min at 4°C. Finally, the samples were centrifuged at 2,000 g for 20 min at 4°C; the supernatants were removed, and the pellets were submitted to radiation counting . The total amount of bradykinin released was calculated, and the data are presented as the mean ± SD, n = 3.A radioimmunoassay was performed to measure bradykinin release according to the procedure described previously . Briefly363-R371 sequence in H-kininogen), and the complex was detected by chemiluminescence. Each figure is representative of the results from three experiments conducted in duplicate.In addition to bradykinin release measured by radioimmunoassay, investigations were conducted to analyze the structure of H-kininogen present in the incubation buffer of CHO-K1 and CHO-745 cells. Briefly, a 35-mm plate containing confluent CHO-K1 or CHO-745 cells was incubated with intact H-kininogen (200 nM) in a single volume of HEPES-Tyrode pH 7.35, without BSA, and in the absence of kininase inhibitors. At the end of a specific time point between 0 min and 3 h, an aliquot of the incubation buffer was removed and mixed with electrophoresis (SDS-PAGE) sample buffer containing the reducing agent β-mercaptoethanol. Following the procedure described below, immunoblotting studies were performed using rabbit IgG anti-H-kininogen (recognizes whole kininogen molecule). After detection by chemiluminescence, the first antigen-antibody complex was removed. The same PVDF membrane was incubated with mouse IgG anti-bradykinin antibody  diluted in serum-free F12 medium with 50 μM Zn2+ for 1 h at 37°C. The biotin-H-kininogen was removed by aspiration, and the cells were fixed with 2.0% paraformaldehyde in 10 mM sodium phosphate and 150 mM NaCl pH 7.4 (PBS). The cells were subsequently permeabilized with PBS containing 0.01% saponin and 1.0% BSA at room temperature. Rabbit IgG anti-caveolin-1 was added at room temperature, and donkey IgG anti-rabbit IgG-FITC was added at room temperature; the biotin-H-kininogen detection was performed with streptavidin-Texas Red conjugated at room temperature. Cells were incubated with DAPI and mounted on glass slides with Fluoromount-G diluted in PBS .Immunocytochemistry experiments were performed in fixed cells, and the H-kininogen was biotinylated as reported previously . CHO-K1 363-R371 sequence in H-kininogen), cells were first washed with serum-free F12 medium with 50 μM Zn2+ and were incubated with LysoTracker Red DND-99 (500 nM) for 20 min diluted in serum-free F12 medium plus 50 μM Zn2 +. Alternatively, cells were pretreated for 24 h with 50 mM sodium chlorate diluted in medium with 50 μM Zn2+. After the addition of LysoTracker Red DND-99, H-kininogen (200 nM) was added in the absence or presence of serine protease inhibitors  diluted in serum-free F12 medium plus 50 μM Zn2+ for 3 h at 37°C. The H-kininogen was removed by aspiration, and the cells were fixed and permeabilized as described above. The mouse IgG anti-bradykinin (recognizes R363-R371 sequence in H-kininogen) was added for 1 h, followed by the addition of goat IgG anti-mouse FITC-conjugated antibody at room temperature. The cells were incubated with DAPI and mounted on glass slides with Fluoromount-G. The cells were examined using a scanning confocal microscope.In experiments in which the H-kininogen was detected by mouse IgG anti-bradykinin  or bradykinin-free H-kininogen-Alexa 488 (80 nM) diluted in serum-free F12 medium plus 50 μM Zn2+. The fluorescent signals of LT Red DND-99 and Alexa 488 (green) as well as the phase contrast micrographs were monitored in real time at 37°C with a confocal laser scanning microscope every 5 min. Each figure is representative of the results from three experiments conducted in duplicate.The endocytosis experiments were performed in real time at 37°C; H-kininogen-Alexa 488 and bradykinin-free H-kininogen-Alexa 488 were prepared as described previously . Briefly2 laser with excitation at 633 nm, and fluorescence emission was detected at 640–670 nm (red channel). Nuclear compartments were stained with DAPI and visualized under 359 nm excitation from a multiphoton laser, and emission was detected at 450–470 nm. Cells were also imaged by differential interference contrast microscopy (DIC) using a HeNe2 laser at 633 nm. False color fluorescent images and transmitted light images (1080 × 1080 pixels) were stored, quantified and managed using the resident LSM Image Browser software . This allowed fluorescence intensity profiles and colocalization rates to be measured, thereby providing mean (± SD) intensity values for the green and red emission channels of each image.The labeled molecules in acidic compartments were analyzed using an inverted confocal laser-scanning microscope . Donkey IgG anti-rabbit IgG-FITC conjugated and goat IgG anti-mouse FITC-conjugated antibodies were visualized under a 488-nm excitation from a CW Argon ion laser, and its emission was detected at 490–550 nm (green channel). LT Red and streptavidin-Texas Red conjugated antibodies were observed with a HeNe2 flasks to confluence followed by incubation with 3.73 mg/mL pancreatin. The cell suspension was added to the culture medium, the solution was collected in a tube, and the supernatant was discarded after centrifugation. Buffer solution  was added to the cell pellet, and the number of cells was counted. The cells were washed two more times with the same buffer solution at pH 5.5 or pH 7.4, and chilled buffer (5.0 ml) was added. The cells were ruptured using a vertical sonicator (3 cycles/30 seconds each cycle and 30 seconds stop between each sonication), and the cell lysates were centrifuged at 9,000 g for 30 min at 4°C. The lysate fraction was separated and stored at -80°C.In the preparation of lysate fractions, CHO-K1 cells were grown in 75 cmAntipain is an inhibitor that blocks the activity of both serine and cysteine proteases . An antiThe protein concentration was determined by the Bradford method using Coomassie Blue dye G250. In the studies of H-kininogen hydrolysis at different time course, a single volume of sample was prepared by maintaining the proportion of intact H-kininogen (1.0 mg) to CHO-K1 lysate fraction (3.5 mg). All samples were incubated in 50.0 mM sodium acetate, pH 5.5 or 20.0 mM HEPES, 5.0 mM NaCl, pH 7.4 at 37°C. At predetermined terms, an aliquot of incubation sample was withdrawn, and the reactions were stopped with sample buffer for electrophoresis and frozen immediately.363-R371 sequence in H-kininogen) or mouse IgG anti-kininogenD6 (recognizes S543-M554 sequence in H-kininogen domain6). Each figure is representative of the results from three experiments conducted in duplicate.The separation of proteins was performed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions . After tP-value of 0.05 or less as statistically significant.The results are shown as mean ± SEM and the statistically significant differences were estimated using analysis of variance and Dunnett’s multiple comparison test accepting a 6 cells) the levels in the absence of kininase inhibitors (1.144 ± 0.040 ng/106 cells); in the presence of serine protease inhibitors, bradykinin release was very low (0.079 ± 0.001 ng/106 cells), indicating that bradykinin release was blocked by 97%. However, in these cells, bradykinin release in the presence of cysteine protease inhibitors was not blocked (2.570 ± 0.210 ng/106 cells). Nevertheless, under the same conditions but at pH 5.5, almost no bradykinin release was detected in the incubation buffer of these cells (data not shown).In this work, we present the results of kinin release in incubation buffer measured by radioimmunoassay after H-kininogen incubation on the surface of CHO-745 cells, which express reduced levels of glycosaminoglycans. The amount of bradykinin in the incubation buffer at pH 7.35 in the presence of kininase inhibitors was two-fold higher than (2.290 ± 0.085 ng/10P < 0.05) and the 62 kDa fragment in size increased significantly ; at the end of incubation (3 h) the H-kininogen 115 kDa in size remained uncleaved but decreased significantly  and the 62 kDa fragment in size increased significantly . In 363-R371 sequence in H-kininogen), a 115 kDa band was observed corresponding to the intact H-kininogen containing the bradykinin domain. However, the 62 kDa band detected by the polyclonal anti-H-kininogen antibody was not identified by the monoclonal anti-bradykinin antibody. In P < 0.0001) and the 62 kDa fragment in size increased but no significantly ; at the end of incubation (3 h) the H-kininogen 115 kDa in size remained uncleaved but decreased significantly  and the 62 kDa fragment in size increased significantly . Similarly, The same experiment was performed at pH 7.35, and the structure of H-kininogen in the incubation buffer of CHO-745 and CHO-K1 cells at different time points was verified by immunoblotting; the results are presented in Confluent CHO-K1 or CHO-745 cells in 35-mm plates were incubated with intact H-kininogen (200 nM) in a single volume of HEPES-Tyrode pH 7.35, without BSA, in the absence of kininases inhibitors. At the end of a specific time point  an aliquot of the incubation buffer was removed and mixed with SDS-PAGE sample buffer containing the reducing agent β-mercaptoethanol. Immunoblot studies were performed using rabbit IgG anti-H-kininogen, and after detection by chemiluminescence, the first antigen-antibody complex was removed. The same PVDF membrane was incubated with mouse IgG anti-bradykinin (recognizes the peptide in H-kininogen R363-R371), and the complex was detected by chemiluminescence. (A) and (B) represent CHO-745 cells developed with rabbit IgG anti-H-kininogen or mouse IgG anti-bradykinin, respectively; (C) and (D) represent CHO-K1 cells developed with rabbit IgG anti-H-kininogen or mouse IgG anti-bradykinin, respectively. The calculated molecular weights (kDa) are presented on the right side of all panels.2+ at 37°C for 20 min. After washing with serum-free F12 medium plus 50 μM Zn2+, the cells were incubated with H-kininogen-Alexa 488 (80 nM) diluted in serum-free F12 medium plus 50 μM Zn2+. The fluorescent signals of LT DND-99 (red) and Alexa 488 (green) as well as the phase contrast micrographs were monitored in real time at 37°C with a confocal laser scanning microscope every 5 min and the figures correspond to 30 min of endocytosis. Endocytosis by CHO-K1 cells in the presence of zinc: H-kininogen-Alexa 488 (A), lysosomes/endosomes labeled with LT Red DND-99 (B), merged images and diphasic contrast (C); absence of H-kininogen endocytosis by CHO-745 cells in the presence of zinc: H-kininogen-Alexa 488 (D), lysosomes/endosomes labeled with LT Red DND-99 (E), merged images and diphasic contrast (F). The orange-yellow signals are indicative of colocalization.Lysosomes/endosomes of living CHO-K1 or CHO-745 cells grown on coverslips were labeled with 0.5 μM LT Red DND-99 in serum-free F12 medium with 50 μM ZnHowever, 2+ at 37°C for 20 min. After washing with serum-free F12 medium plus 50 μM Zn2+, the cells were incubated with bradykinin-free H-kininogen-Alexa 488 (80 nM) diluted in serum-free F12 medium plus 50 μM Zn2+. The fluorescent signals of LT DND-99 (red) and Alexa 488 (green) as well as the phase contrast micrographs were monitored in real time at 37°C with a confocal laser scanning microscope every 5 min and the figures correspond to 30 min of endocytosis. Normal endocytosis: (A) bradykinin-free H-kininogen-Alexa 488, (B) lysosomes/endosomes labeled with LT Red DND-99, (C) merged images and diphasic contrast. The orange-yellow signals are indicative of colocalization.Lysosomes/endosomes of living CHO-K1 cells grown on coverslips were labeled with 0.5 μM LT Red DND-99 in serum-free F12 medium with 50 μM Zn2 +. The involvement of caveola in H-kininogen endocytosis by CHO-K1 was evaluated in serum-free F12 medium with 50 μM Zn2+ for 1 h at 37°C. The cells were fixed and permeabilized, and rabbit IgG anti-caveolin-1 was added. Detection was achieved using a donkey IgG anti-rabbit IgG-FITC conjugated antibody. The biotin-H-kininogen detection was achieved with a streptavidin-Texas Red conjugated antibody. (A-C) Colocalization of H-kininogen and caveolin in CHO-K1 cells: (A) caveolin detected by rabbit IgG anti-caveolin-1 and donkey IgG anti-rabbit IgG-FITC conjugated antibodies (green); (B) biotin-H-kininogen developed with a streptavidin-Texas Red conjugated (red); and (C) composite of images presented in (A) and (B). (D-F) Colocalization of H-kininogen and caveolin in CHO-745 cells: (D) caveolin detected by rabbit IgG anti-caveolin-1 and donkey IgG anti-rabbit IgG-FITC conjugated antibodies (green); (E) biotin-H-kininogen developed with a streptavidin-Texas Red conjugated (red); and (F) composite of images presented in (D) and (E). The orange-yellow signals are indicative of colocalization.CHO-K1 or CHO-745 cells grown on coverslips were incubated with biotin-H-kininogen (200 nM) diluted in serum-free F12 medium plus 50 μM Zn363-R371 sequence in H-kininogen). The endocytosis of H-kininogen, containing the bradykinin domain, was evaluated by mouse IgG anti-bradykinin (recognizes R2 + for 20 min to label lysosomes/endosomes. CHO-K1 cells were pretreated or not for 24 h with sodium chlorate (50 mM) diluted in medium plus 50 μM Zn2+. After LT Red DND-99 treatment, H-kininogen (200 nM) was added in the absence or presence of serine protease inhibitors  diluted in serum-free F12 medium plus 50 μM Zn2+ for 3 h at 37°C. The cells were fixed and permeabilized, and bradykinin was detected by mouse IgG anti-bradykinin (recognizes the peptide in H-kininogen R363-R371) complexed to goat IgG anti-mouse FITC-conjugated antibody. The cells were examined by confocal fluorescence microscopy. The endocytosis and intracellular localization of H-kininogen containing the bradykinin domain (green) are indicated by colocalization with acidic vesicles previously labeled with LT Red DND-99 (red). Normal endocytosis by CHO-K1: H-kininogen containing bradykinin (A), lysosomes/endosomes labeled with LT Red DND-99 (B), and merged images (C); CHO-K1 cells treated with sodium chlorate: H-kininogen containing bradykinin (D), lysosomes/endosomes labeled with LT Red DND-99 (E), and merged images (F); CHO-K1 cells in presence of serine protease inhibitors: H-kininogen containing bradykinin (G), lysosomes/endosomes labeled with LT Red DND-99 (H), and merged images (I); and absence of H-kininogen containing bradykinin endocytosis by CHO-745 cells: H-kininogen containing bradykinin (J), lysosomes/endosomes labeled with LT Red DND-99 (K), and merged images (L). The orange-yellow signals are indicative of colocalization and the nucleus was stained in blue with DAPI.CHO-K1 or CHO-745 cells were grown on coverslips and incubated with LT Red DND-99 (0.5 μM) in serum-free F12 medium plus 50 μM ZnThe lysate fraction of CHO-K1 cells was prepared after sonication as described in the Material and Methods section. A single volume of sample was prepared, maintaining the proportion of intact H-kininogen (1.0 mg) to CHO-K1 lysate fraction (3.5 mg). All samples were incubated in buffer at pH 7.4 or pH 5.5 at 37°C. At predetermined times , an aliquot of incubation sample was withdrawn, and the reactions were stopped with sample buffer for SDS-PAGE under reducing conditions and frozen immediately. After the protein was electroblotted onto PVDF membranes, a primary antibody and a secondary antibody were used. The antigen-antibody complex was developed with the SuperSignal chemiluminescent substrate. (A) HEPES 20.0 mM, NaCl 5.0 mM, pH 7.4 and (B) sodium acetate 50.0 mM, pH 5.5. Control sample “C”: H-kininogen (1.0 μg) 4 h incubation in the buffer. The calculated molecular weights (kDa) are presented on the right side.543-M554 sequence in H-kininogen domain6). 543-M554 (D6 domain). The pH of the buffer did not influence the activity of either lysate fraction, retained and not retained on antipain-Sepharose, and both lysate fractions differed from each other with respect to bradykinin release.In an attempt to separate the enzymes present in the lysate fraction prepared from CHO-K1, antipain was used as a ligand in affinity chromatography. The fractions retained or not retained on the resin were tested for their kininogenase activity. The analysis of H-kininogen or its fragments was accomplished by switching antibodies after stripping the same membrane. The experiment was performed in triplicate. To confirm the results, the order of addition and removal of antibodies was switched. The antibodies used included rabbit IgG anti-H-kininogen, mouse IgG anti-bradykinin and mouse IgG anti-kininogenD6 (recognizes S543-M554 sequence in H-kininogen domain6). Control sample “C”: H-kininogen (1. 0 μg). The calculated molecular weights (kDa) are presented on the right side.H-kininogen (1.0 μg) was incubated at pH 5.5 or pH 7.4 with lysate fractions from CHO-K1 cells (4.0 μg), retained or not on antipain-Sepharose, for 4 h at 37°C. The proteins under reducing conditions were separated by 13% SDS-PAGE. A primary antibody and secondary antibody were used, and the antigen-antibody complex was developed with the SuperSignal chemiluminescent substrate. In an attempt to characterize the fragments of H-kininogen, different primary antibodies were used after the first development by stripping the antigen-antibody complex from the PVDF membrane according to the Methods section. (A) the first antibody is rabbit IgG anti-H-kininogen; (B) the first antibody is mouse IgG anti-bradykinin (recognizes the peptide in H-kininogen R363-R371); and (C) the first antibody is mouse IgG anti-kininogenD6  in CHO-K1 after 3 h incubation . In the These findings suggest the role of cysteine proteases in H-kininogen processing on the cell surface and in acidic endosomal vesicles. The affinity chromatography using antipain-Sepharose of the CHO-K1 lysate fraction was performed and successfully separated kininogenases. There was no difference in the assays at pH 5.5 and 7.4, but the proteins in the fraction bound to resin released bradykinin from H-kininogen, whereas the proteins in the fraction unbound to resin cleaved H-kininogen at other sites but did not release bradykinin .Taken together, the data in the present work support the hypothesis that after blood vessel injury, H-kininogen can interact and bind to the surface of non-endothelial cells. On lipid raft domains/caveolae or endosomes, PGs mediate the endocytosis of intact H-kininogen, which can be processed by different classes of proteases  43]. Th. Th43].


Tumour vasculature is generally disordered because of the production of excessive angiogenic factors by tumour cells, which results in tumour progression and reduces the effectiveness of radiotherapy or chemotherapy. Transient anti-angiogenic therapies that regulate tumour vascular morphology and function and improve the efficiency of antitumour therapy are under investigation. Recombinant human endostatin (Endostar/rhES) is a vascular angiogenesis–disrupting agent that has been used to treat non-small cell lung cancer (NSCLC) in the clinical setting. In this study, we used gold nanoparticles (AuNPs) as a drug-delivery system (DDS) for targeted tumour delivery of rhES for short therapy, which resulted in transient tumour vascular normalization, reduced permeability and hypoxia, strengthened blood vessel integrity, and increased blood-flow perfusion. Moreover, combination therapy with 5-FU over this timeframe was substantially more effective than 5-FU monotherapy. In conclusion, our research demonstrates the potential use of AuNPs as a drug-delivery platform for transporting rhES into a tumour to induce transient tumour vascular normalization and enhance the antitumour efficacy of cytotoxic drugs. Gold nanoparticles (AuNPs), as a nanoparticle DDS, exhibit unique properties, including chemical stability, inherent biocompatibility, low cytotoxicity1620To improve In this study, we used AuNPs as a drug carrier to deliver rhES into a tumour via passive targeting and facilitate transient vascular normalization to improve antitumour therapeutic efficacy.The UV-visible spectrum of AuNPs revealed a characteristic surface plasmon resonance (SPR) absorption band at approximately 517 nm; after combination with rhES, the compound exhibited a peak at 550 nm, which was attributed to the introduction of groups on the surface of the AuNPs . DynamicP = 0.0091) and 7.4 (P = 0.0040)  continuously increased with time, and the release capability was increased gradually with the reduction of the pH value (pH 6.6 and 6.2). Thus, low pH resulted in more rapid release of rhES compared with release at pH 6.8 ( 0.0040) . The linP = 0.0141) and 4 h (P = 0.0332)  . The difin vivo imaging of Cy5-labeled regents revealed that after the i.v. injection, rhES-AuNPs-PEG rapidly accumulated in tumours as early as 30 min and increased in tumours in 4 h  as a marker of pericytes, and FITC-labelled dextran as a marker of permeability. CD31 staining revealed that the vessels in the control treatment were chaotic and deformed . By contP = 0.0009; day 8, P = 0.0011; day 12, P = 0.2206; P = 0.0078; day 8, P = 0.0082; day 12, P = 0.0815; The control tumours exhibited increased vascular permeability, which is associated with excessive deposition of dextran around the tumour vessels, as well as irregular CD31 staining. However, after treatment with rhES-AuNPs-PEG for 4 days and 8 days, leakage of dextran from vessels decreased , which enables tumour cells to produce a variety of pro-angiogenic factorsP = 0.0428; P = 0.8948; P = 0.1166; To determine if vessel normalization increases chemotherapeutic efficacy, we administered 5-FU within and outside this normalization window. Compared with monotherapy, combination with rhES-AuNPs-PEG significantly increased 5-FU localization to the tumour sections (P = 0.0012), and this advantage continued until day 24 (P = 0.0025). This result is consistent with the results of the HPLC analysis, which revealed high concentrations of 5-FU in tumours after pre-treatment with rhES-AuNPs-PEG. The median survival time of mice treated with rhES-AuNPs-PEG plus 5-FU was 1.60-fold longer than that of the placebo group , thus targeting delivery into tumours more efficiently. As previously mentioned, extravasation of nanoparticles, as a passive targeting method, is dependent on heterogeneous vesselsEndostatin is a broad-spectrum endogenous angiogenesis inhibitor2732Pericytes have been implicated as mediators of tumour angiogenesis and metastasis35343637in vitroHypoxia is an indicative feature of solid tumours and a potent inducer of tumour angiogenesis3942Next, we examined the concentration of 5-FU in tumours by HPLC analysis. Treatment with rhES-AuNPs-PEG increased the concentration of intratumour 5-FU, which is closely related to increased blood flow in tumours, and this improvement was obvious after 4 and 8 days of treatment with rhES-AuNPs-PEG. Moreover, 5-FU combined with tumour vascular normalization therapy reduced disease progression.In summary, we prepared a tumour-specific targeting drug-loaded nanosystem by surface-modifying rhES using AuNPs. Our data indicate that AuNPs enhanced the concentration of rhES in tumours. Furthermore, rhES promoted transient vascular normalization in H22 xenograft models, reduced hypoxia and leaking, and improved pericyte coverage and blood perfusion, thereby increasing 5-FU delivery into the tumour and improving therapeutic efficiency. However, further research is needed to fully elucidate the mechanism of tumour vascular normalization.2.rhES was provided by Simcere-Medgenn Bio-pharmaceutical Co., Ltd.  and stored at 4 °C. 5-FU was purchased from Shanghai Xudong Haipu Pharmaceutical Co., Ltd. AuNPs were obtained from Shanghai Jie Ning Biotech Co., Ltd. The AuNPs exhibited an average diameter of 15 nm and were dissolved in distilled water and stored in a light-resistant container at 4 °C. Methoxy polyethylene glycol-Thiol  was purchased from Shanghai zzbio Co., Ltd . The H22 cell line was purchased from Sun Yat-sen University, cultured in DMEM  containing 10% foetal bovine serum  and 1% penicillin/streptomycin (Hyclone), and maintained in a 37 °C incubator with 5% COin vivo treatment. SPR in the UV-visible spectrum was observed on a model V-570 Jasco dual-beam spectrophotometer. The hydrodynamic size distribution and zeta potential were evaluated by DLS using a Malvern Zetasizer Nano ZS . The morphology and surface structure were visualized using field emission transmission electron microscopy (FETEM) .The rhES-AuNPs-PEG was prepared via a self-assembly process. A calculated amount of rhES was added to the dispersion of AuNPs, and the reaction system was incubated in the dark at 4 °C for 12 hours. The preparation was oscillated gently every 2 hours. Subsequently, mPEG-Thiol was introduced and incubated for another 4 h for The rhES-load efficiency was determined according to the previously described method® Ultra, 30-kD molecular weight cutoff). rhES-AuNPs-PEG and 10-mL PBS at different pH values were added to the filter device and centrifuge tube, respectively. The system was incubated at 37 °C for a predetermined length of time, and the filter device was centrifuged at 3000 rpm for 20 min. Next, 1 mL of PBS was withdrawn and replaced by an equal volume of fresh PBS. The rhES concentration was determined as described previously.rhES release behaviour from the carrier was estimated using a centrifugal filter device  were purchased from the Medical Laboratory Animal Center  and maintained under specific pathogen-free conditions. All animal study protocols were approved and conducted in accordance with the guidelines of the Laboratory Animal Ethics Committee of Jinan University.6 H22 cells into the subcutaneous tissue of the right flank. The tumour volume was measured using a Vernier calliper every other day and calculated as V = (length × width2) × 0.523. Treatment with rhES-AuNPs-PEG or placebo was initiated when the tumour volume reached 175–200 mm3 post-inoculation and was repeated daily from day 0 to day 12. From day 1, all animals bearing tumours underwent treatment with 5-FU (40 mg/kg) daily for 4 days, followed by treatment every 2 days for 1 week. Subsequently, the mice were euthanized, and the tumours and various organs were harvested for analysis.Xenografted tumours were initiated by injection of 5 × 10in vivo imaging systems  to detect the rhES-AuNPs-PEG biodistribution. The animals were anaesthetized with isoflurane and imaged 10 min, 30 min and 4 h after the injection of Cy5-labelled regents. The acquisition time was approximately 10 min, and image reconstruction was performed using InVivoScope 1.37 software (Bioscan).Mice bearing H22 tumours received rhES (10 mg/kg) or rhES-AuNPs-PEG containing the same dose of conjugated rhES for predetermined times (30 min and 4 h). The animals were sacrificed, and tissue samples from the liver, spleen, heart, lung, kidney, and tumours were collected and rapidly frozen for immunofluorescence detection using an anti-endostatin antibody . In addition, we used optical + area to CD31+ area using Image J software.Extravascular diffusion visualization was assessed by intravenous injection of 0.25 mg/mouse FITC-labelled dextran . Tumours were harvested after 20 minutes, frozen in optimal cutting temperature compound  and stored at −80 °C. Cryosections with a thickness of 20 μm were fixed in cold acetone and rehydrated in PBS. Microvessels were stained with an anti-CD31 antibody , and the percentage of leakage was calculated as the ratio of the dextran+ area was presented as a percentage of the CD31+ area.Tumour blood perfusion was detected using FITC-conjugated lectin from Bandeiraea simplicifolia  and anti-CD31 staining. Subsequently, 10 mg/kg FITC-lectin was injected intravenously. After 1 minute, the tumours were excised, and sections were prepared as described previously. Tissue was visualized under a fluorescence microscope (Leica DM6000B), and the lectin+ vessels. The ratio of hypoxia in the tumour sections was presented as HIF-1α staining area/CD31+ area.Tissues were fixed in 4% paraformaldehyde for 24 hours and paraffin-embedded, sectioned, dewaxed in xylene, and rehydrated through graded alcohols. Antigen retrieval was performed in citric acid buffer (pH 6.0). Sections were blocked in 2% normal goat serum for 1 hour and stained with primary antibodies as follows: anti-CD31 antibody  for endothelium, α-SMA antibody  for pericytes, and HIF-1α antibody  for hypoxia. The sections were then washed and incubated with rhodamine-conjugated goat anti-rat IgG (H + L)  or goat anti-rabbit IgG-FITC  for 40 min at RT. The level of pericyte coverage was presented as a percent of the length along CD31® Technologies, Santa Clara, CA, USA) on a COSMOSIL C18 column  . The eluents were methanol, 0.1% trifluoroacetic acid and deionized water , and the flow rate was 1.0 mL/min. 5-FU was detected at a wavelength of 265 nm. All responses obtained were analysed using Agilent® ChemStation® software.The concentration of 5-FU in different organs was quantified using an Agilent 1100 series HPLC . P values less than 0.05 were considered statistically significant .All data are presented as the mean ± standard error of the mean of independent triplicate samples. Statistics for different groups were compared by one-way analysis of variance or unpaired Student’s How to cite this article: Li, W. et al. Gold Nanoparticle-Mediated Targeted Delivery of Recombinant Human Endostatin Normalizes Tumour Vasculature and Improves Cancer Therapy. Sci. Rep.6, 30619; doi: 10.1038/srep30619 (2016).


Drug delivery using focused ultrasound  has attracted significant interest during the last few years. Focused Ultrasound (FUS) can induce local tissue hyperthermia, increasing blood flow and vascular permeability, which then enhances the uptake of therapeutics by target tissues. Although a number of studies have focused on nanoparticles (e.g. thermosensitive liposomes) little work has been done on the effects of FUS on the uptake of macromolecular drugs into tumours. In this study we aim to understand the effect of FUS-induced hyperthermia on tumour vascular permeability, as measured by the increased uptake of labelled macromolecules in the tumour. Using optical imaging we monitor the labelled macromolecules tumour localisation in real time in both short (minutes to hours) and long (weeks) term.Albumin (Murine Serum MSA), IgG, and Trastuzumab were labelled using a NIRF  and a coupling protocol developed in house. The NIRF labelled macromolecules were purified using a PD-10 column and were characterised by HPLC and SDS-PAGE (both unheated and after heating to 42°C for 7 min). SCID mice were used to form tumours using IGROV-1 (ovarian cancer cells) injected s.c. to both flanks. When tumours reached suitable size (~6 mm diameter) animals were injected (i.v. tail vein) with the labelled antibodies. FUS applications were performed post injection, on the right tumour using an Ultrasound Therapy Imaging Probe System .FUS insonations induced a localised temperature increase which was monitored using fine-wire thermocouples inserted above and below the tumour body. Temperature was kept at 42±0.3°C for 2-5 min. Multispectral optical imaging was used to assess the accumulation of labelled macromolecules in tumours over time with or without FUS-induced hyperthermia.in vivo, FUS induced hyperthermia enhanced significantly their uptake by the tumours. Figure shows the effect of FUS induced hyperthermia on murine serum albumin tumour uptake. FUS treatments induced immediate increase on macromolecular drug uptake by tumours. The macromolecules remained in the tumour for several days . Trastuzumab appeared to persist in the tumours for a longer period probably due to its specificity for Her-2 receptors. The effect of this rapid and tumour localised macromolecular drug concentration can lead to an improvement of the therapeutic effect, this still needs to be investigated. Image guided antibody drug delivery using FUS can become a useful imaging and therapeutic tool.For all three macromolecules tested


Glioblastoma multiforme (GBM) is the most common tumour of the central nervous system in adults and while it is not frequently considered a paediatric malignancies, it is in terms of 'years of life lost' among the most devastating cancers in children and adolescences. The current standard of care consists of tumour resection followed by radiotherapy and a course of Temozolomide. However, despite intense efforts, the mean patient survival of 12-15 months has not significantly improved over the last two decades.While the PI3K/Akt/mTOR 'survival' pathway is aberrantly activated in almost 90% of GBMs and despite promising pre-clinical results, pharmacological inhibitors of this signalling cascade have so far not fulfilled their clinical potential.In this study we use different cell populations cultured directly from primary-derived patient material  to investigate the combination of a pharmacological inhibitor of PI3K, GDC-0941, and clinically relevant doses of radiation. While the induction of apoptosis and the retardation of proliferation is enhanced by the combination treatment in a statistically significant manner, the overall effect is lacklustre. However, importantly, the enhanced motility induced in differentiated GBM cells after radiation can be blocked by PI3K inhibition, while neither radiation, nor GDC-0941 had an effect on the motility of the highly motile cancer stem cells. These data suggest that the role of Pi3K-mediated signalling and the primary mode of movement difference in genetically identical, but epigenetically different GBM cells. We therefore propose that the key role of PI3K inhibitors in combination therapy might lie not in their ability to enhance apoptosis induction, but in preventing tumour invasion in a subpopulation of tumour cells.


Dynamic social media content, such as Twitter messages, can be used to examine individuals’ beliefs and perceptions. By analyzing Twitter messages, this study examines how Twitter users exchanged and recognized toponyms (city names) for different cities in the United States. The frequency and variety of city names found in their online conversations were used to identify the unique spatiotemporal patterns of “geographical awareness” for Twitter users. A new analytic method, Knowledge Discovery in Cyberspace for Geographical Awareness (KDCGA), is introduced to help identify the dynamic spatiotemporal patterns of geographic awareness among social media conversations. Twitter data were collected across 50 U.S. cities. Thousands of city names around the world were extracted from a large volume of Twitter messages (over 5 million tweets) by using the Twitter Application Programming Interface (APIs) and Python language computer programs. The percentages of distant city names (cities located in distant states or other countries far away from the locations of Twitter users) were used to estimate the level of global geographical awareness for Twitter users in each U.S. city. A Global awareness index (GAI) was developed to quantify the level of geographical awareness of Twitter users from within the same city. Our findings are that: (1) the level of geographical awareness varies depending on when and where Twitter messages are posted, yet Twitter users from big cities are more aware of the names of international cities or distant US cities than users from mid-size cities; (2) Twitter users have an increased awareness of other city names far away from their home city during holiday seasons; and (3) Twitter users are more aware of nearby city names than distant city names, and more aware of big city names rather than small city names. Internet-based human communication and messaging has rapidly expanded due to social networking sites (SNS) where people continue to share their ideas, images, news, memes and advertisements at a prodigious and increasing rate. SNS  dynamically produce huge quantities of instant messages that often reveal the locations of their users and the locations where the messages were created. The locations of users can be found in their user profiles, if they disclose this information to the public. In some cases, global positioning systems (GPS) in mobile phones are used to identify the latitude and longitude where the messages were created, known as geotags. Social media data with geolocation tags or location profiles are being increasingly used in geographical research projects, and in the emerging business of location based services –7. HowevThis research aims to employ the names of cities mentioned in Twitter messages to understand Twitter users’ geographical awareness. A new analytic method, Knowledge Discovery in Cyberspace for Geographical Awareness (KDCGA), has been developed to facilitate the discovery of geographical knowledge from large volumes of social media data. In developing the KDCGA, about 5 million Twitter messages were collected across 50 cities in the U.S. over three months. Thousands of city names throughout the world were identified from the Twitter messages (tweets). The frequency of mentioning of city names was quantified and used as an element to estimate the level of geographical awareness for Twitter users living in the same city. This estimation enables a comparison of the spatial and temporal change of geographical awareness for Twitter users from different U. S. cities over time.Quantifying the level of geographical awareness is a key element in this study. We introduce the global awareness index (GAI) to quantify the level of geographical awareness of Twitter users from the same city. A high GAI indicates that Twitter users from the same city mention international city names, or distant U.S. city names, much more than nearby local city names. Conversely, a low GAI indicates that the frequency of international or distant city names mentioned in the messages is lower than the frequency of local city names mentioned. In short, Twitter users who mention faraway city names more frequently have a higher GAI—i.e., levels of geographical awareness. We propose that the GAI as developed in this study can be used to quantify the level of globalization worldwide. Many researchers have attempted to quantify the level of globalization in the form of indices and argued the importance of quantification –11. One Everything is related to everything else, but near things are more related than distant things.” [One goal of this study is to test Tobler’s first law of geography in cyberspace. The law states: “things.” . If Toblthings.” , 14–i.e.In what cities do Twitter users have high levels of geographical awareness? It is hypothesized that Twitter users in the largest cities  are more aware of distant city names and international city names than those who live in mid-size cities .How does the geographical awareness of Twitter users change over time? What differences in the temporal level of geographical awareness characterize Twitter users from different regions? It is hypothesized that Twitter users tend to mention far-away city names from their residence more frequently during holidays than during regular days.What are the spatial patterns of geographical awareness for Twitter users from the same or different cities? It is hypothesized that Twitter users are likely to mention closer cities than distant cities. However, they might be more likely to be more aware of big city names rather than small city names.The research is intended to address the following three questions:Social media applications such as Twitter and Facebook have recently gained a spectacular popularity. According to Twitter, over 500 million active Twitter users produced about 350 million messages per day in 2013. To exploit these massive social media data bases, extensive studies have been conducted to develop human knowledge, including analyzing human communication and networks –17, sentCognitive geography focuses on the study of how the human mind perceives and interacts with its geographical surroundings through the senses. Cognitive geography is based on the idea that individuals apply learned geographic knowledge about the spatial and non-spatial characteristics of their environment. The knowledge is gained, processed and stored in the human mental system . The orget al. [Some studies have attempted to evaluate an individual’s level of cognition or awareness about space and places , 32. Recet al.  analyzedIn the current study, the act of naming a place within one’s Twitter messages is assumed to imply awareness of that name. This does not necessarily mean that the Twitter users can pinpoint the exact location of the cities or describe detailed information about the cities. For example, if a user tweets, “I am going to Chicago for the annual meeting this year,” the user already knows or is aware of the city of Chicago. But we cannot tell if users have sufficient geographic knowledge about Chicago, such as the exact location of Chicago or the total population of Chicago. Geographical awareness is different from geographic knowledge. Care must be taken to distinguish among different cities with the same name  and between dissimilar semantics .Social media research has increasingly focused on the analysis of big data , 34, 35.Twitter is an Internet-based social networking and microblogging service that permits registered users to receive and broadcast very short text messages (tweets), up to 140 characters in length. Users and collective sites are identified by user names and hashtag identifiers such as “#katyperry”. Messages can be resent or forwarded (retweeted), and groups of associates can associate together (as friends) in sending and receiving group tweets. References to Internet URLs are made by compression, for example into tinyurls.To collect and analyze the spatial and temporal patterns of Twitter messages, two steps are involved. The first step is to collect tweets using Twitter Application Programming Interfaces (APIs) , which aIn this study, tweets containing the names of cities all over the world were collected within a 20 mile buffer from the center of each of 50 cities in the U.S. . These cFor geocoding processes, place name gazetteers were provided by the GeoNet Names server, supported by the National Geospatial Intelligence Agency (earth-info.nga.mil/gns/html). This geographical gazetteer database covers all countries and contains over eight million place names in multiple languages. This gazetteer is useful for geocoding since it provides the coordinates of all cities in the world. The challenges in geocoding are discussed in detail in the methodology section below.Focusing on big data analysis of social media messages, this study adopted the research framework of Knowledge Discovery in Cyberspace (KDC) . KDC is Based on the original framework of KDC, this study introduced a new method, called Knowledge Discovery in Cyberspace for Geographical Awareness (KDCGA) . KDCGA iThe first step of KDCGA is to query tweets using the Twitter Search APIs with keywords being the list of city names. Twitter Search APIs provide their own query functions that allow us to query tweets including defined keywords. The original Twitter data has many elements such as the text of the messages, screen name, profile background url, followers count, friends count, and hashtag. When the query function provided by Twitter Search API is used, the program queries tweets containing the city names anywhere in all of these elements. Therefore, above all, the program removes tweets that do not contain the keywords (city names) in the text of the Twitter messages. After this process, only those tweets that contain the keywords in the text remain.A major procedure in the first step is to define the keywords (city names) for collecting tweets. A problem in deciding keywords is that many cities in the world share the same name, and that some place names are words in another context. To analyze distributional patterns of tweets containing city names, these tweets are supposed to be geocoded in the later step. In terms of geocoding, if all city names in the world were unique, it would be straightforward to match each city name contained in the tweets with the coordinate of each city provided by the gazetteer. However, in reality, there are two or more different cities that have identical names , which cTo prevent confusion, our program only queries tweets containing both names of cities and names of the states or country that the cities belong to. This has the advantage of also eliminating the non-toponyms, such as Jack London. The second step is a process of advanced filtering. This process aims to eliminate the tweets that do not contain actual city names as much as possible. The keywords used in the Twitter search APIs need to follow predefined Boolean operations and query string formats. Some city names as keywords can create unexpected query results. For example, if we search tweets with the keyword, “Athens, Georgia”, the Twitter query function would search any tweets containing the two individual keywords: “Athens”, and “Georgia”. In other words, the query function selects all tweets containing those two words anywhere in the twitter data regardless of whether they are uppercase or lowercase, together or separated. One possible tweet of an unexpected query result that does not contain an actual city name could be “Now the Korean drama, the battle of athens, is available at Zion market, Georgia” This tweet contain the city name, “athens” and state name, “Georgia”, but the words in the tweet actually do not mean the city of Athens in the state of Georgia. In the advanced filtering process, the program removes the tweets that do not contain a city name and a state name next to each other or a city name and a country name or code next to each other if the city is the outside the U.S. As long as these words are positioned together, the city names are valid regardless of whether they are uppercase or lowercase. For example, city names in tweets such as “San Diego, California”, “California san diego”, “Athens of Greece” and “Athens in Georgia” are acceptable.The last two steps are for the visualization and spatial analysis of the tweets collected in the previous steps. The third step is to match each city name mentioned in the tweet with the geographic coordinates of the city, and to visualize them as points on the map. The coordinates of each city are available from gazetteers provided by the GeoNames gazetteer. If city names were mentioned multiple times in a single tweet, each city name was geocoded and represented as a dot on the map.x and y coordinates of all points, representing the overall location of points. The mean center is influenced by outliers far away from the major clusters, and must be converted from spherical to linear distance. The median center is another way to represent the overall location of points by identifying the location that minimizes overall Euclidean distance to the points in a dataset. The median center is less influenced by outliers than the mean center. The standard deviational ellipse summarizes the spatial characteristic of a set of geographic features and provides metrics of central tendency, dispersion and directional trend [Step four consists of a set of various spatial analyses to estimate the level of geographical awareness. To examine the difference of spatial distribution of city names mentioned in the tweets in different regions, three centrographic measures–mean center, median center and standard deviational ellipse—were adopted from previous work . The meaal trend .n is the total number of keywords (cities mentioned in tweets) at one of the 50 home cities in Xi is the number of tweets containing a particular keyword (city name). Di is a dimensionless value to indicate the distance between the center of one of home cities and the center of a city mentioned in tweets. To calculate Di, the actual distance is calculated and divided by the half length of earth’s circumference (the maximum possible city separation). Great-circle distance was used to calculate the distance between the two places on the globe. p is the number of people resident in each of the 50 home cities according to the U.S Census Bureau. Dividing by p facilitates a fair comparison of the level of geographical awareness of Twitter users in the different 50 home cities.The global awareness index (GAI) was created to compare the differences in the levels of geographical awareness of Twitter users living in different regions. In this study, GAI at each of the 50 cities was measured and compared. The mathematical expression for calculating GAI can be given as:The global awareness index (GAI) changes based on the distance, the size of home city, and the number of city names mentioned in tweets. GAI increases as the distance between a home city and the cities mentioned in tweets increases. GAI also increases as the number of cities mentioned in tweets increase. However, GAI decrease as the size of home city increases. A high GAI means that users frequently mention cities that are far away from their residence compared to nearby cities. If Twitter users in region A mention more distant cities than users in region B and the number of population is the same in both cities, region A will have a higher GAI than region B.x, y, z and outputs a map in raster format where each cell has a value representing the level of intensity. Concentrations of intense values are hotspots. In this study, per Twitter users living in each home city  and z (the frequency of each city mentioned) at one of home cities in x, y, z values of tweets created by Twitter users in another home city. This method was used to compare the difference of the level of geographical awareness of Twitter users between pairs of home cities.Kernel density estimation (KDE) is used to identify the hotspots of tweets. KDE has been used to map the distribution of social media data in many studies—e.g., , 42, 43.ome city , KDE inpx axis) and the global awareness index (GAI) of Twitter users (y axis). The Pearson correlation coefficient (r) was used to reveal this relationship because it measures the strength and direction of the linear relationship. The correlation coefficient between the two variables is 0.52, which means that there is a weak positive relationship between the two variables. In other words, the level of geographical awareness (i.e. GAI) is likely to be high in cities with high populations. Also the top 10 highest population cities mostly show fairly high GAIs. Therefore, the general trend is that users living in highly urbanized areas tend to mention far away cities rather than nearby cities in their tweets compared to those in less urbanized areas. This trend implies that big cities have more frequent movement of people, ideas, and commodities between faraway cities than small cities.The level of geographical awareness of Twitter users varies depending on where the messages were created. The GAI quantifies the level of geographical awareness and enables comparison of the level of geographical awareness of Twitter users living in different cities in the U.S. The spatial difference of the geographical awareness can be examined by mapping and using spatial analyses. GAI reveals the arithmetic difference in the geographical awareness of Twitter users living in different cities. On the other hand, mapping and spatial analyses enable the visualization of the distributional patterns of the geographical awareness. For example, in Figs There is a clear distributional difference in the awareness of global cities between Twitter users living in New York (NY) and those in Los Angeles (LA). NY and LA were chosen for the comparison because these cities are highly populated and their GAIs are similar—the GAI in NY is 0.76, and in LA is 0.73 . First, The level of geographical awareness of Twitter users in the U.S. tends to be high at the end of the calendar year. Twitter users are more aware of cities that are close to their home than more distant cities in accordance with Tobler’s First Law of Geography. Twitter users are generally more aware of nearby cities than distant cities. However, if cities mentioned in tweets are far away from the city where they live, they are more aware of big cities rather than small cities. This research analyzed Twitter messages to identify spatiotemporal differences in the level of geographical awareness of Twitter users living in various regions across the U.S. KDCGA, consisting of four steps  led to the discovery of the following new facts. First, Twitter users living in heavily populated areas have more geographical awareness of cities that are far away than those living in less populated areas. This is an indication of a hierarchical effect in global awareness, the so-called global or world cities have far longer reach than other cities. Second, twitter users are more aware of more distant cities during the holiday season in late December than the remainder of the year. This maybe because of travel planning, gift giving or just staying in touch with distant family and friends. It is possible that other holiday events such as the Muslim Haj, the Chinese lunar new year celebration and the U.S. Spring break show similar patterns. Third, Twitter users are much more aware of nearby cities than distant cities, as predicted by Tobler’s first law of geography. This leads us to conclude that the first law of geography applies equally to both fixed geographic space and the cyberspace of internet-based social media. However, the distance decay of Tobler’s law appears to apply only at one level of the urban hierarchy, such that the largest distance major cities can appear to be virtually closer than smaller nearby cities. Thus to a New Yorker, London England may seem closer than Albany, the state capital just 250km away.The findings in this study show an effect of distance decay in terms of recognizing city names of Twitter users. Distance decay describes the decline of human interactions between places with increasing distance from their point of origin . The effThe level of geographical awareness is not only based on distance, but also on a model of spatial interaction, the gravity model. The gravity model assumes that the attraction or interaction between two cities is proportional to the product of their sizes and inversely proportional to their separation distance . The shoThe pattern of geographical awareness of Twitter users demonstrates a particular pattern of spreading ideas called hierarchical diffusion. “Hierarchical diffusion refers to the hopscotch spread from larger, more social dominant centers to smaller ones, typically along the national or international transport network” , 46. ForThe GAI can potentially be used to estimate the relative degree of globalization of cities. Various methods for measuring the level of globalization with a composite index have been discussed—e.g., –11. The The use of social media messages provides a cost effective way to estimate a dynamically changing level of globalization. Globalization indices developed in previous research are computed based on the aggregation of multiple variables revealing cultural, ecological, economic, political and social aspects of countries , 48. TheWe acknowledge that publicly available tweets are only a sample of all tweets, and that only a few of them carry a geotag that allows verification that the user is at their stated location, or one of the places mentioned in the text. We further recognize that culling all tweet geography from what is usually transient and fickle information exchanges may reveal more about human intent and interest that actual travel or communication desires. Nevertheless, there is abundant opportunity to learn more about geography from the large numbers of exchanges now enabled by location-aware social media.S1 TableColumn A is each 50 city represented in the map of (XLSX)Click here for additional data file.S2 TableColumn A is a list of city names mentioned by Twitter users living in San Jose, California. Column B represents state code or country code where the respective city in column A belongs. Column C represents state name or country name where the respective city in column A belongs. Column D and E represent coordinate of each city. F column shows the population in each city. G column represents the number of tweets mentioned each city.(XLSX)Click here for additional data file.S3 TableColumn A is a list of city names mentioned by Twitter users living in Jacksonville, Florida. Column B represents state code or country code where the respective city in column A belongs. Column C represents state name or country name where the respective city in column A belongs. Column D and E represent coordinate of each city. F column shows the population in each city. G column represents the number of tweets mentioned each city.(XLSX)Click here for additional data file.S4 TableColumn A is a list of international city names mentioned by Twitter users living in NY. Column B represents state code or country code where the respective city in column A belongs. Column C represents state name or country name where the respective city in column A belongs. Column D and E represent coordinate of each city. F column shows the population in each city. G column represents the number of tweets mentioned each city.(XLSX)Click here for additional data file.S5 TableColumn A is a list of international city names mentioned by Twitter users living in LA. Column B represents state code or country code where the respective city in column A belongs. Column C represents state name or country name where the respective city in column A belongs. Column D and E represent coordinate of each city. F column shows the population in each city. G column represents the number of tweets mentioned each city.(XLSX)Click here for additional data file.S6 TableColumn A is a list of US city names mentioned by Twitter users living in NY. Column B represents state code where the respective city in column A belongs. Column C represents state name where the respective city in column A belongs. Column D and E represent coordinate of each city. F column shows the population in each city. G column represents the number of tweets mentioned each city.(XLSX)Click here for additional data file.S7 TableColumn A is a list of US city names mentioned by Twitter users living in LA. Column B represents state code where the respective city in column A belongs. Column C represents state name where the respective city in column A belongs. Column D and E represent coordinate of each city. F column shows the population in each city. G column represents the number of tweets mentioned each city.(XLSX)Click here for additional data file.S8 TableColumn A is a list of US city names mentioned by Twitter users living in Chicago. Column B represents state code where the respective city in column A belongs. Column C represents state name where the respective city in column A belongs. Column D and E represent coordinate of each city. F column shows the population in each city. G column represents the number of tweets mentioned each city.(XLSX)Click here for additional data file.S9 TableColumn A is a list of US city names mentioned by Twitter users living in Boston. Column B represents state code where the respective city in column A belongs. Column C represents state name where the respective city in column A belongs. Column D and E represent coordinate of each city. F column shows the population in each city. G column represents the number of tweets mentioned each city.(XLSX)Click here for additional data file.S10 TableColumn A is a list of US city names mentioned by Twitter users living in Charlotte. Column B represents state code where the respective city in column A belongs. Column C represents state name where the respective city in column A belongs. Column D and E represent coordinate of each city. F column shows the population in each city. G column represents the number of tweets mentioned each city.(XLSX)Click here for additional data file.S11 TableColumn A is a list of US city names mentioned by Twitter users living in Houston. Column B represents state code where the respective city in column A belongs. Column C represents state name where the respective city in column A belongs. Column D and E represent coordinate of each city. F column shows the population in each city. G column represents the number of tweets mentioned each city.(XLSX)Click here for additional data file.


Background: Determination of oocyte fertilization and embryo quality are one of the most important purposes in ART cycles. Follicular fluid provides an important microenvironment for development of oocytes and some biochemical characteristics of the follicular fluid, such as pregnancy-associated plasma protein-A (PAPP-A), may play an important role in prediction of success rate of ART. Objective: This study was performed to evaluate whether there was any difference in follicular fluid PAPP-A, fertilization, and embryo quality between GnRH agonist long protocol and flexible GnRH antagonist multiple-dose protocol in ART cycles.Materials and Methods: A total of 100 women who were candidates for ART were enrolled the study and were divided into two groups, GnRH agonist (GnRHa) long protocol (n=51) and flexible GnRH antagonist (GnRHant) multiple-dose protocol (n=49). Follicular fluid sample was obtained from a single mature follicle and follicular fluid PAPP-A level, fertilization and embryo quality of the same oocyte were evaluated in both groups.Results: There was no significant difference in the mean levels of follicular fluid PAPP-A between the GnRHa protocol and GnRHant protocol . The mean levels of follicular fluid PAPP-A in fertilized oocyte and good quality embryo were comparable in GnRHa and GnRHant protocols.Conclusion: Our data indicated that no differences of follicular fluid PAPP-A levels were observed between cycles using GnRHa long protocol and those of using flexible GnRHant multiple-dose protocol. Ovarian follicular development is an essential factor for success rate of assisted reproductive technology (ART). Determination of fertilized oocyte and embryo quality are one of the most important purposes in ART cycles -4.In recent years, different ovarian stimulation protocols have been used to increase fertilized oocyte and good quality embryos. GnRH agonists (GnRHa) and GnRH antagonists (GnRHant) are two controlled ovarian stimulation (COS) methods that have widely used for in vitro fertilization (IVF) and embryo transfer (ET) cycles , 5-7. ThFollicular fluid (FF) provides an important microenvironment for development of oocyte and some biochemical characteristics of the follicular fluid surrounding the oocyte may play a critical role in determining the oocyte quality, the subsequent fertilization and embryo development. Thus follicular fluid content may play an important role in prediction of success rate of ART , 11-13. 2, thus indirectly enhances FSH action on the ovary . Patients were divided into two groups, GnRHa long protocol and flexible GnRHant multiple-dose protocol.COS protocols2 ≤50 pg/ml and the absence of ovarian cyst by using transvaginal ultrasound, the buserelin dose was reduced to 0.25 mg/day and was continued until the day of HCG injection. Ovarian stimulation was initiated with HMG  150 IU/day on the day 2 of menstrual cycle.In GnRHa long protocol (n=51), pituitary down-regulation was done with SC injection of 0.5 mg/day buserelin  which was started on day 21 of the previous menstrual cycle. After confirmation of pituitary suppression, using serum EIn flexible GnRHant multiple-dose protocol (n=49), ovarian stimulation was performed by administration of HMG 150 IU/day on the day 2 of menstrual cycle without previous oral contraceptive pretreatment and when at least one follicle by mean diameter ≥14 mm diagnosis, 0.25 mg GnRH anagonist  SC daily was started and was continued until the day of HCG injection. 2 levels and gonadotropin dosage was adjusted according to individual's response and was continued until the day of HCG injection and when at least two follicles reached a mean diameter of 18 mm, 10000 IU HCG  was applied IM and oocyte retrieval was performed 34-36 hours after HCG injection.In both groups, ovarian response was monitored by both transvaginal ultrasound and serum ECollection of follicular fluidSingle follicle with a diameter greater than 17 mm was obtained individually during oocyte retrieval and contaminated follicular fluid with blood was excluded the study. Immediately after single follicular aspiration, the cumulus oocyte complex was separated from FF and then FF was centrifuged for 15 min and centrifuged FF was stored at -80°c until the samples were all completed. Also the obtained single oocyte was cultured in a separated culture dish and ICSI was performed on that oocyte 4-6 hours after oocyte retrieval. Fertilization of the same oocyte was evaluated 16-18 hours after ICSI and fertilized oocyte was defined as zygote with two pronucleous (2PN). Quality of embryo was assessed on fertilized oocyte 48 hours after ICSI and was scored based on the shape, number and fragmentation of blastomers  and embrStatistical analysist-test or chi-square test where appropriate. P-value less than 0.05 was considered to be statistically significant.The parameters were analyzed with the SPSS version 15.0 . The results were compared between the two groups and statistically analyzed using the Student’s There was no significant difference between two protocols in regard to basic characteristics' of patients . EtiologThe mean levels of follicular fluid PAPP-A with good quality embryo were comparable in GnRHa and GnRHant protocols  . There wOverall, a clear correspondence between specific follicular fluid biochemical characteristics, and fertilization and embryo development has not been established in the literature. It appears that ovarian folliculogenesis requires dynamic interactions between mature oocytes, granulose cells and regulatory factors present in the follicular fluid and suitable follicular environment is necessary for good oocyte quality , 5.In the present study, the mean levels of follicular fluid PAPP-A were similar in GnRHa long protocol and flexible GnRHant multiple-dose protocol  and we found any correlation between FF levels of PAPP-A, and fertilization and good quality embryo in GnRHa protocol compared to those of GnRHant protocol.et al , but intrafollicular PAPP-A dose not substantially elevate the PAPP-A serum concentration and it seems PAPP-A acts only locally and cleaves IGFBP-4 within the ovarian follicle (Despite many advantages on the clinical results of COH, the role of GnRHa and GnRHant protocols on folliculogenesis and its effect on intrafollicular microenvironment remains controversial , 28-30. f PAPP-A . Moos etfollicle .IGFs are stimulating steroidgenesis and IGFBPs are effective antigonadotropins. Both of them play an essential role on follicular growth. IGFBP-4 is an inhibitor of IGF activity and FSH stimulated granulose steroidgenesis and PAPP-A, by cleaving IGFBP-4, is providing higher IGF-II activity in the follicles , 31-33.et al showed that the mean levels of follicular fluid PAPP-A in GnRHa long protocol was 0.53±0.25 mIU/ml compared to 0.50±0.31 mIU/ml in GnRHant protocol (There are few studies in regard to follicular fluid PAPP-A between the two protocols, GnRHa and GnRHant protocols , 11, 24.In present study, we evaluated FF concentration of PAPP-A, while there are several endocrine/paracrine and ovarian local regulatory factors that may be have positive or negative effects in IVF-ET cycles. Although remarkable progress has been made in understanding the biology of PAPP-A during recent years, further studies is needed to evaluate other underlying mechanisms or factors that are involved in fertilization and embryo development in IVF-ET cycles. Our data indicated that no differences of follicular fluid PAPP-A levels were observed between cycles using GnRHa long protocol and those of using flexible GnRHant multiple-dose protocol and it appears follicular fluid PAPP-A cannot be considered as a marker of fertilization and embryo quality in IVF-ET cycles that are used GnRHa or GnRHant protocols.


Hemorrhagic shock resuscitation aims at maintaining tissue perfusion while waiting for hemorrhage control. Fluid resuscitation remains the first line therapy to improve tissue perfusion but a vasoconstrictor (most often norepinephrine) may be associated to stabilize the mean arterial pressure (MAP) level and avoid hemodilution while waiting for transfusion. However, the arteriolar vasoconstriction induced by norepinephrine could have deleterious effects on tissue perfusion during hypovolemia due to hemorrhage.To evaluate the effects of norepinephrine administration on intestinal perfusion and oxygenation during hemorrhagic shock resuscitation in mice.th and the 120th minute: a fluid resuscitated group (FR) with NaCl 0,9% to reach a MAP level of 60 mmHg and a group resuscitated with fluid and norepinephrine (FRNE) to reach a MAP level of 60 mmHg. Mice were then retransfused with their shed blood and an equal volume of Ringer Lactate and were observed between the 120th and the 165th minute. Intestinal microcirculation was observed by intravital microscopy. Intestinal mucosal PO2 level (PO2muq) was measured by phosphorescence quenching (OxyMicro probe) at jejunal mucosal level. A sham group  was constituted. Data are presented as mean ± SEM. The group effect on microcirculatory and oxygenation parameters was analysed with ANOVA and Mann Whitney tests.Tracheotomised and ventilated Balb/c mice were submitted to a controlled hemorrhagic shock to reach a MAP level of 40 mmHg during sixty minutes. Mice groups differed from their resuscitation between the 602muq from 33,9 ± 3 to 8,2 ± 1 mmHg  and from 32,9 ± 2 to 10,3 ± 2 mmHg  was observed during hemorrhage in FR and FRNE groups respectively. During hemorrhagic shock resuscitation, MAP goal was reached in FRNE group (59 ± 1 mmHg) but not in FR group (52 ± 2 mmHg). Fluid resuscitation amount was 67 ± 14 µL.g-1 and 176 ± 19 µL.g-1 in FRNE and FR groups respectively (p < 0.05). The alteration of intestinal microcirculatory perfusion was corrected in the same proportion in FRNE and FR groups. The PO2muq recovered to its basal level during resuscitation in the FR group: 30 ± 3 mmHg and in the FRNE group: 33 ± 2 mmHg.Hemorrhagic shock induced an alteration of the intestinal microcirculatory perfusion with a decrease in the fraction of perfused villi and a decrease in the villous red blood cells flux. A decrease of the POIn a mice model of uncontrolled hemorrhagic shock, a MAP directed resuscitation associating norepinephrine and fluid resuscitation decreased blood loss and fluid requirements compared to a MAP directed resuscitation with fluid without norepinephrine while preserving intestinal microcirculatory perfusion and oxygenation.


In the Funding section, the grant from the Institute of Medical Microbiology and Immunobiology, University of Szeged, Hungary by the Hungarian Government is listed incorrectly. The correct grant number is: TÁMOP-4.2.2.A-11-1-KONV-2012-0035.


Hemangioblastoma is a benign cerebellar tumour which may occur as a sporadic entity or in association with von Hippel-Lindau (VHL) disease in approximately 25% of cases. Renal hemangioblastoma (RH) is an extremely rare and newly recognised tumour. Here, we describe five cases of RH, one discovered by CT in an accident and the other four detected during routine examinations.Five cases of renal hemangioblastoma retrieved from the Department of Pathology, Fudan University Shanghai Cancer Center were studied and the literatures were reviewed. Immunohistochemistry was used to differentiate and confirm this tumour.Pathological examination following tumour resection revealed RH in all cases, the first patient was also diagnosed with renal cell carcinoma (RCC), suggesting the possibility of VHL syndrome, but PCR sequencing analysis of the VHL gene confirmed no mutation in any of the three exons, implying sporadic disease .Histologically, the tumours were circumscribed, composed of sheets of oval or polygonal cells and a prominent vascular network. Tumour cells had pleomorphic nuclei, but mitotic figures were rare. The diagnosis of hemangioblastoma was confirmed by immunohistochemistry.RH is very rare and is challenging to differentially diagnose. Distinguishing RCC and RH is difficult and each has a different prognosis, so differentiating between them is essential for avoiding over-diagnosis and unnecessary treatment. Hemangioblastoma is a benign tumour of uncertain histogenesis characterised by the presence of stromal cells and a rich vascular component.3Hemangioblastoma has also been reported outside the CNS, for example in the peripheral nerves,All cases were sent to one of the authors for consultation. H&E-stained sections (4 mm thickness) were re-examined to evaluate the tumour's histological features and immunohistochemistry was performed with an avidin-biotin-complex immunoperoxidase technique. Antibody details are given Clinical demographics and follow-up data were obtained from medical records and the referring physicians. The institutional review board for human studies of Fudan University Shanghai Cancer Center approved this retrospective study. Patient records or information were anonymised and de-identified prior to analysis. Written informed consent was provided by participants or their next-of-kin for their clinical records to be used in this study.VHL gene confirmed no mutation in any of the three exons, suggesting sporadic disease. The patient died before the next follow-up.A 30-year-old man was admitted to the hospital for examination after an accident and a CT scan revealed a mass with a different density within a right kidney lesion. The malignancy was subsequently imaged by MRI. Surgical resection of the tumour revealed a collision tumour comprising RCC and extraneural hemangioblastoma, among which RCC accounts for less than 5% of the total tumour volume. The mass involved the renal cortex and medulla and measured 3.2×2.5×1.4 cm. VHL disease was initially suspected. However, PCR sequencing analysis of the A CT scan during a routine examination revealed that a 57-year-old woman had a mass in her right kidney. A radical nephrectomy was performed as a malignant tumour was suspected. H&E slides confirmed the diagnosis of RCC. No surgery or chemotherapy was carried out. The patient is currently alive with no evidence of recurrent tumour or the development of other tumours.The patient was a 48-year-old man with no hereditary diseases. When he visited the hospital for evaluation of organ function, abdominal sonography revealed a tumour mass in the right kidney. A CT scan confirmed a 2.3 cm mass with heterogeneous density in the lower portion of the right kidney. As RCC was suspected, the patient underwent a right radical nephrectomy. There was no evidence of tumour recurrence 42 months after the surgery. Follow-up magnetic resonance brain imaging did not any reveal tumours.A 25-year-old man was confirmed to have a left renal tumour during a routine examination, and was admitted to hospital for urological examination. The patient was asymptomatic with a normal appetite, no abdominal pain, no weight changes, and no family history of renal disease. CT imaging revealed a 3.6 cm well-defined, round, heterogeneously enhancing mass in the left kidney. Nephrectomy was performed and a 4.1 cm encapsulated tumour removed. Follow-up at 27 months confirmed no tumour recurrence or metastasis.A previously healthy 36-year-old woman presented with a tumour on the left kidney during a routine examination. The tumour was an unencapsulated but sharply circumscribed nodule. Clear cell RCC (CCRCC) was diagnosed. Immunohistochemistry of a tissue sample suggested a diagnosis of RH. The patient was alive 3 months later.Microscopically, most tumours were well circumscribed and well demarcated from the surrounding renal parenchyma; only one tumour had areas of poorly marginated growth. The RHs all consisted of sheets of oval or polygonal cells traversed by a prominent, arborising vascular network A, B. TheMost cases had similar immunohistochemical profiles. Tumour cells expressed inhibin (3/5) and vimentin (3/5) in 60% of cases A, B. NeuHemangioblastoma is a benign tumour of uncertain histogenesis that generally occurs in a relatively restricted area of the CNS.VHL disease is an autosomal dominant hereditary disorder characterised by retinal and CNS hemangioblastomas, pheochromocytoma and CCRCC.The tumours in these five cases were morphologically similar to lesions arising in the CNS, which are also composed of sheets of oval or polygonal cells with palely eosinophilic and clear microvacuolated cytoplasm, separated by a delicate capillary network, and usually with interspersed larger thin-walled and thick-walled blood vessels.Compared with CNS hemangioblastoma, the results of immunohistochemical staining reported by many authors do not suggest a specific line of differentiation for RH tumours.In the current study, S100 and NSE appeared to be the most sensitive markers and were expressed in all cases, as was inhibin. Vimentin expression was observed in 60% of cases. Of note, expression of inhibin was less in our cases than reported in the RH literature; sample limitations may explain this discrepancy. The fact that vimentin is strongly expressed in these tumours has led investigators to suggest they have an undifferentiated mesenchymal origin.Extraneural renal hemangioblastoma (RH) is a rare, newly recognised tumour with morphological features similar to its cerebellar counterpart.This report describes the morphological features and immunohistochemical characteristics of five cases of RH and summarises the findings.RH is frequently misdiagnosed, so immunohistochemistry is essential for differentiating RH from morphologically similar neoplasms, such as renal cell carcinoma.RH is frequently misdiagnosed as RCC, and is likely to be under-recognised as it mimics many tumour types morphologically and is usually not considered in a differential diagnosis.


Tumours can be categorised based on their stromal architecture into tumour vessel and stromal vessel phenotypes, and the phenotypes have been suggested to define tumour response to chronic treatment with a VEGFR2 antibody. However, it is unclear whether the vascular phenotypes of tumours associate with acute vascular response to VEGFR tyrosine kinase inhibitors (TKI), or whether the early changes in vascular function are associated with subsequent changes in tumour size. This study was sought to address these questions by using xenograft models of human non-small cell lung cancer (NSCLC) representing stromal vessel phenotype  and tumour vessel phenotype , respectively.For dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI), nude mice bearing established Calu-3 or Calu-6 xenografts were treated with a potent pan-VEGFR TKI, cediranib (6 mg/kg), at 0 h and 22 h. DCE-MRI was performed 2 h before the first dose and 2 h after the second dose of cediranib to examine acute changes in tumour vessel perfusion. Tumours were harvested for hypoxia detection by CA9 immunohistochemistry. For tumour growth study, mice carrying established Calu-3 or Calu-6 tumours were treated with cediranib once daily for 5 days.Twenty-four hours after cediranib administration, the perfusion of Calu-3 tumours was markedly reduced, with a significant increase in hypoxia. In contrast, neither perfusion nor hypoxia was significantly affected in Calu-6 tumours. Tumour regressions were induced in Calu-3 xenografts, but not in Calu-6 xenografts, although there was a trend towards tumour growth inhibition after 5 days of cediranib treatment.These findings suggest that tumour stromal architecture may associate with acute tumour vascular response to VEGFR TKI, and this acute tumour vascular response may be a promising early predictive marker of response to VEGFR TKI in NSCLC. Angiogenesis is essential for tumour growth, invasion, and metastasis by supplying nutrients and oxygen As anti-angiogenic treatment-induced changes in tumour vascularity occur ahead of the reduction in tumour size, measurement of functional changes in tumour vessels may identify early response to anti-angiogenic treatment. Dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) is a non-invasive imaging modality that can detect changes in tumour perfusion, and it has been used to assess anti-vascular therapy response in both preclinical and clinical studies Based on stromal architecture, it has been proposed that human tumours can be categorised into two phenotypes: the tumour vessel phenotype in which blood vessels are distributed amongst tumour cells, and the stromal vessel phenotype in which blood vessels are embedded in stroma 22.1n = 38; adenocarcinoma (n = 14), squamous cell carcinoma (n = 24)) were obtained from ProteoGenex, Inc.  with appropriate ethical approval and with informed consent.Formalin-fixed, paraffin embedded NSCLC tumour samples  were subcutaneously injected at a single site on the back of the mouse. Mouse weights and tumour volumes were measured three times per week (volume = 1/2 × length × width × depth).All animal experiments were performed under a licence issued under the UK Animals (Scientific Procedures) Act of 1986. To establish xenografts, female BALB/C nude mice  were anaesthetised with 2% isoflurane and 5 × 102.43, mice were randomised into two groups. For DCE-MRI study, mice (n = 3/group) were dosed with either cediranib  6 mg/kg or vehicle 1% polysorbate by oral gavage at 0 h and 22 h. Tumours were imaged by DCE-MRI 2 h before the first treatment and 2 h after the second treatment of cediranib or vehicle. Animals were sacrificed immediately after the second DCE-MRI. For tumour growth study, mice (n = 3/group) were treated with either cediranib or vehicle 1% polysorbate once daily for 5 days. Tumour volumes were measured over the course of treatment.When tumours reached approximately 150 mm2.5MRI was performed in a 4.7T horizontal magnet  using a 25 mm quadrature birdcage coil. Respiration-gated 3D gradient echo imaging  covering a field of view of 54 × 27 × 27 mm at an isotropic resolution of 0.42 mm was used for the DCE. T1 was quantitatively mapped using variable flip angles nominally in the range of 0.5–8 degrees with RF transmission field inhomogeneities accounted for The DCE-MRI signal was converted to gadolinium concentration as described 2.6Sections (4 μm) from paraffin-embedded tumours from xenografts or NSCLC tumour samples were deparaffinised followed by heat-induced epitope retrieval in citrate buffer (pH6.0). Sections were stained for CA9 (carbonic anhydrase 9) or CD31 using Dako EnVision™ - Dual link system-HRP (Dako) according to the manufacturer's instructions. The antibodies used for immunohistochemistry were: CA9  and CD31 . Hematoxylin and eosin (H&E) and Masson's trichrome staining were performed for routine histological examination. Human NSCLC tumours were scored for stromal vessel and tumour vessel phenotypes based on CD31 and Masson's trichrome stains. The presence of intravascular erythrocytes identified functional vessels. Tumours showing strong predominance (≥90%) of functional vessels within desmoplastic stroma or within tumour tissue were defined as either stromal vessel or tumour vessel phenotypes, respectively. Where functional vessels consisted of >10% of each phenotype, the tumours were scored as a mixed vessel phenotype. Similarly, 12 NSCLC xenografts  and an additional 31 xenografts or allografts from other types of tumours  were assessed for vessel phenotypes.2.7P < 0.05.Data were expressed as mean ± SEM. Statistical analysis was performed using Student's t-test by SPSS software. Statistical significance was defined as 33.1Calu-3 and Calu-6 xenografts were stained for the endothelial cell marker CD31. Consistent with the previous report 2 and functional vessels were identified by the presence of intravascular erythrocytes. Among the human NSCLC tumour samples (n = 38), 19 exhibited a stromal vessel phenotype and 18 exhibited a mixed vessel phenotype, with only one sample showing exclusively a tumour vessel phenotype  (P < 0.001), whereas IAUC in Calu-3 tumours was not significantly affected by vehicle treatment (P = 0.727)   A, sugges= 0.727) B. In con= 0.803) C. Calu-3= 0.803) D. Calu-6= 0.803) D. Taken To relate changes in IAUC to histopathology, the extent of necrosis was assessed by H&E staining E. Necros3.3P < 0.001; P = 0.382; To investigate whether the tumour perfusion change resulted in oxygenation changes, tumour hypoxia was assessed by immunohistochemical staining for the endogenous hypoxia marker CA9 3.4P < 0.01) (P = 0.07).To evaluate whether the early reduction in tumour perfusion was associated with tumour growth inhibition, mice bearing Calu-3 and Calu-6 tumours were treated with cediranib once daily for 5 days, and tumour volumes were measured and plotted. Regression of Calu-3 tumours was evident as early as 2 days after the onset of cediranib treatment, and persisted throughout the whole treatment course. Statistically significant growth inhibition by cediranib in Calu-3 tumours was obtained on day 4 post-treatment, and continued till the experiment's end on day 5. ( < 0.01) A. Howeve < 0.01) B, it did4It has been suggested that the vascular phenotypes of tumours categorised by stromal architecture can define tumour response to chronic VEGF-targeted monotherapy Previous gene expression analysis has shown that the two vascular phenotypes of tumours exhibit differential gene expression profiles Acute vascular response is characteristic of vascular disrupting agents (VDAs) Ktrans, a biomarker of tumour perfusion and vessel permeability, after 3 or 5 days of treatment in Calu-6 xenografts, suggesting that more prolonged treatment with cediranib can produce significant anti-vascular effects. Nonetheless, in agreement with our study, no tumour growth inhibition was observed over 5 days of cediranib treatment. This is consistent with other findings whereby significant tumour growth inhibition was only evident in Calu-6 xenografts after 2 weeks of DC101 treatment In contrast, no significant effect of acute cediranib treatment was detected in Calu-6 tumours by using DCE-MRI. It has previously been reported that cediranib-induced vessel pruning was only apparent in the periphery of the Calu-6 tumours Although xenograft models in NSCLC and other cancers exhibit distinct vessel phenotypes, human NSCLC tumours are histologically heterogeneous as shown in our study: a high proportion of tumours having a mixed vessel phenotype, with both tumour vessel and stromal vessel phenotypes present within the same tumour. In addition, in our analysis, only 1/38 human NSCLC tumour samples had a predominantly tumour vessel phenotype compared with 10/12 NSCLC xenograft models. This suggests that the tumour vasculature in xenograft models is not a good representation of the clinical situation. Therefore, defining patient subgroups by tumour stromal architecture can be difficult in the clinic, especially where only small biopsies are available.Of interest, Calu-3 tumours were better and more uniformly perfused throughout the tumour than the Calu-6 xenografts which were well-perfused only at the tumour rim, suggesting that DCE-MRI may provide a potential non-invasive biomarker to discriminate stromal and tumour vessel phenotypes. It has been reported that acute changes in tumour vessel function are more predictive of tumour growth response than changes in histology or gene profiles It is noteworthy that reduced perfusion can lead to acute hypoxia as shown in Calu-3 tumours. Increased hypoxia might decrease the efficacy of cytotoxic therapies when anti-VEGF/VEGFR treatment is combined with radiotherapy or chemotherapy. However, some studies have shown that even in the case of tumour hypoxia induced by antiangiogenic therapy, the antitumour effects can still be enhanced by combined chemotherapy In this study, acute vascular response to cediranib was evaluated in two NSCLC xenograft models. Future studies are warranted to evaluate a broad range of xenograft models in lung cancer and other tumour types. Furthermore, it will be of interest to determine the correlation between the acute vascular response to VEGFR TKI and the long-term outcome in NSCLC patients.In conclusion, we propose that tumour stromal architecture may affect the response of tumour vasculature to VEGFR TKI treatment, and that acute change in tumour perfusion may be predictive of tumour response to VEGFR TKI in NSCLC.None declared.