Journal: PLoS ONE

Article Title: Identification of Novel Biomarkers in Seasonal Allergic Rhinitis by Combining Proteomic, Multivariate and Pathway Analysis

doi: 10.1371/journal.pone.0023563

Figure Lengend Snippet: Nasal fluid proteins enriched in the acute phase response pathway .

Article Snippet: Histidine-rich glycoprotein (HRG) was analyzed with an ELISA kit from Cusabio Biotech Co., Ltd (Wuhan, China).

Techniques: Clinical Proteomics

Journal: EMBO Molecular Medicine

Article Title: Histidine-rich glycoprotein modulates neutrophils and thrombolysis-associated hemorrhagic transformation

doi: 10.1038/s44321-024-00117-y

Figure Lengend Snippet: ( A ) Representative images of isolated peripheral blood neutrophils treated with tPA (10 μg/ml), tPA + HRG (1 μM), and LPS (10 μg/ml). Neutrophils were incubated for 2.5 h and co-stained with H3Cit (green), MPO (red), and DAPI (blue). White arrows indicate NET formation. Scale bar = 20 µm. ( B – D ) Statistical analysis and comparison of H3Cit, MPO, and NET formation rates in different groups ( n = 5–8). The results shown are the mean ± SEM. ( E – G ) The concentration of TNF-α ( n = 10), IL-8 ( n = 11), and ROS ( n = 9) in the supernatant of the culture system was quantified by ELISA. Data information: Data are presented as mean ± SEM; two-tailed Kruskal–Wallis H test is used in ( B – G ). .

Article Snippet: Plasma samples were collected from whole blood and HRG levels were measured using a human HRG ELISA kit (KIT10836, Sino Biological, Beijing, China).

Techniques: Isolation, Incubation, Staining, Comparison, Concentration Assay, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: EMBO Molecular Medicine

Article Title: Histidine-rich glycoprotein modulates neutrophils and thrombolysis-associated hemorrhagic transformation

doi: 10.1038/s44321-024-00117-y

Figure Lengend Snippet: Reagents and tools table

Article Snippet: Plasma samples were collected from whole blood and HRG levels were measured using a human HRG ELISA kit (KIT10836, Sino Biological, Beijing, China).

Techniques: Recombinant, Sequencing, In Vivo, SYBR Green Assay, Software, Bradford Assay, Enzyme-linked Immunosorbent Assay, Membrane

Journal: EMBO Molecular Medicine

Article Title: Histidine-rich glycoprotein modulates neutrophils and thrombolysis-associated hemorrhagic transformation

doi: 10.1038/s44321-024-00117-y

Figure Lengend Snippet: Reagents and tools table

Article Snippet: Recombinant histidine-rich glycoprotein , Sino Biological , 10836-H08H.

Techniques: Recombinant, Sequencing, In Vivo, SYBR Green Assay, Software, Bradford Assay, Enzyme-linked Immunosorbent Assay, Membrane

Journal:

Article Title: Histidine-Rich Glycoprotein Modulates the Anti-Angiogenic Effects of Vasculostatin

doi: 10.2353/ajpath.2010.090782

Figure Lengend Snippet: HRGP CLESH domain fusion protein specifically precipitates Vstat120 from tumor cell postculture media. A: HRGP/GST fusion proteins. Three recombinant GST fusion proteins were designed using fragments of the HRGP protein spanning amino acids 155 to 213 (within the second cystatin domain), amino acids 330 to 389 (the HRR), and amino acids 428 to 502 (CLESH domain). The linear structure of CD36 with the CLESH domain highlighted is shown below for reference. B: Coomassie stained gel (top panel) showing the purified GST-HRGP protein fragments after glutathione sepharose chromatography. The bottom panel shows Western blot analysis of each fusion protein probed with anti-GST antibody. C: Western blot analysis of a GST pull-down assay. The three GST-HRGP fusion proteins and GST alone were bound to glutathione sepharose beads, and CM from LN229V120 glioma cells were added to each sample. Each precipitate was then probed using the Vstat120/BAI1-specific antibody. Vstat120 was only detected when the CLESH domain (HRGP 428 to 502) was used in the pull-down and not the cystatin (HRGP 155 to 213) or the HRR (HRGP 330 to 389) domains. HRGP 428 to 502 only precipitated Vstat120 when CM from Vstat120-transfected LN229 cells (+ lanes) was used. No protein was detected when CM from the nontransfected parent cells (− lane) was used. This is a representative image from n = 3 experiments.

Article Snippet: Anti-human HRGP monoclonal antibody (MAB1869) was purchased from R&D Systems (Minneapolis, MN) and anti-human N-terminal BAI1 polyclonal antibody was previously described.

Techniques: Recombinant, Staining, Purification, Chromatography, Western Blot, Pull Down Assay, Transfection

Journal:

Article Title: Histidine-Rich Glycoprotein Modulates the Anti-Angiogenic Effects of Vasculostatin

doi: 10.2353/ajpath.2010.090782

Figure Lengend Snippet: LN229V120 glioma cloned cell lines stably express HRGP. A: Western blot of CM from 13 different cloned HRGP LN229V120 glioma lines probed with antibodies to HRGP (top panel) and Vstat120 (bottom panel). Lines 5, 18, 22, and 23 all showed considerable expression of HRGP while retaining Vstat120 expression. The control line (6A8) was transfected with empty vector and expressed Vstat120, but not HRGP as expected. Other lines either expressed Vstat120 alone 8, 20 or neither protein. 9, 16, 19, 25, 28, 29 B: Relative levels of expression of HRGP transfected lines. The amount of CM tested was normalized to total cell number at time of collection. HRGP expression levels in CM were assessed by Western blot and compared with that of line 23. Lines 5 and 23 were almost identical in expression level, whereas line 22 expressed fivefold more compared with the other two (*P < 0.002) C: HRGP expression did not have an effect on in vitro growth rates. All cells were seeded at the same cell density and proliferation was monitored by crystal violet assay. All lines proliferated at near identical rates and reached plateau at days 6 to 7.

Article Snippet: Anti-human HRGP monoclonal antibody (MAB1869) was purchased from R&D Systems (Minneapolis, MN) and anti-human N-terminal BAI1 polyclonal antibody was previously described.

Techniques: Clone Assay, Stable Transfection, Western Blot, Expressing, Control, Transfection, Plasmid Preparation, In Vitro, Crystal Violet Assay

Journal:

Article Title: Histidine-Rich Glycoprotein Modulates the Anti-Angiogenic Effects of Vasculostatin

doi: 10.2353/ajpath.2010.090782

Figure Lengend Snippet: HRGP reverses inhibition of endothelial cell migration and tube formation by Vstat120. A: Scratch assays. A scratch was made within a confluent monolayer of HDMVECs and the cells were incubated overnight with normal media (media only – lane 1) showing moderate migratory growth into the acellular area of about 40% total closure. When CM from Vstat120-expressing cells (6A8 – lane 7 and LN229Vstat120 – lane 3) was added, fewer cells were seen migrating from the original cell boundary, especially when compared with the parental LN229 line (lane 2). An average of 30.3% and 31.7% closure, respectively, was calculated for the Vstat120-rich CMs used versus 57% for LN229. When CM from lines expressing both Vstat120 and HRGP (line 5 – lane 4, line 22 – lane 5, and line 23 – lane 6) was added cell migration equal to or greater than cells treated with normal growth media only was seen and approximately doubled that of the 6A8 control. The percent wound was 65.1%, 64.6%, and 56.0% for the three HRGP lines compared with 30.3% for the 6A8 control (*P < 0.003 for all cases). B–F: Matrigel tube formation assays. Equal numbers of HDMVECs were seeded onto a matrigel support and incubated overnight with Vstat120-only CM (LN229Vstat120 or the HRGP control line 6A8), normal growth media (media only), or CM from three lines expressing both Vstat120 and HRGP (lines 5, 22, and 23.) With no CM added, after 14 hours some cells have begun to spread and form tube-like structures with an average of 12.7 branch points per 10× field (F – lane 1 – media only; 2 fields in each of 3 separate experiments were counted). This is reduced when Vstat120 is present in the original Vstat120 cell line or in the HRGP control line (6A8 – E) with 6.8 and 7.3 branch points counted for each (F – lanes 3 and 7). On addition of HRGP (B–D), increased numbers of web-like structures are observed consistent with tube formation. The average number of branch points was 18.2, 17.3, and 17.1 for the three HRGP lines 5, 22, and 23, respectively (*P < 0.002 when compared with the control 6A8 line). HRGP attenuates Vstat120-induced apoptosis. G: Western blot of caspase-3 activation of HDMVECs after treatment with CMs containing either Vstat120 or Vstat120 and HRGP and quantification of caspase-3 activation (H). CMs with Vstat120 show a 2.5-fold (HRGP control 6A8) and fourfold (LN229Vstat120) increase in caspase-3 activation over serum-free media. When HRGP is present with Vstat120, caspase-3 activity returns to basal levels (*P < 0.04 when comparing HRGP lines 5, 22, and 23 to the control line 6A8).

Article Snippet: Anti-human HRGP monoclonal antibody (MAB1869) was purchased from R&D Systems (Minneapolis, MN) and anti-human N-terminal BAI1 polyclonal antibody was previously described.

Techniques: Inhibition, Migration, Incubation, Expressing, Control, Western Blot, Activation Assay, Activity Assay

Journal:

Article Title: Histidine-Rich Glycoprotein Modulates the Anti-Angiogenic Effects of Vasculostatin

doi: 10.2353/ajpath.2010.090782

Figure Lengend Snippet: HRGP increases tumor growth and vascularity in Vstat120-expressing glioma cells transplanted subcutaneously into nude mice. A: Cloned cells that stably express both HRGP and Vstat120 (lines 5, 22, and 23) or Vstat120 alone (line 6A8) were injected subcutaneously into immunocompromised mice (n = 8 tumors per group) and tumor volumes were calculated over a seven-week period. Line 22, which expresses approximately 5× the amount of HRGP compared with lines 5 and 23, showed a rapid increase in tumor size from day 43 until sacrificed at day 50 due to tumor burden (*P < 0.0001 when comparing line 22 to control line 6A8). B–F: Increased vascularity in HRGP-expressing glioma tumors. Tumors from animals described in A were removed and paraffin sections processed and stained with monoclonal anti-vWF or control antiserum. Slides were developed with an HRP-conjugated avidin-biotin staining system and counterstained with hematoxylin. Matched regions of the tumors were used for vessel analysis. Black arrows show vWF-positive vessels stained brown from the DAB substrate. A representative field of a Vstat120-only expressing tumor (6A8, B) showed no apparent staining when control serum was added. The same field stained with vWF on an adjacent section showed small spread out vessels (C) that are quantified in F. HRGP-expressing tumor line 5 (D) and line 23 showed similar staining patterns to the Vstat120-only tumor (6A8), but HRGP line 22 shows a marked increase in vessel density and size when compared with the other lines (E). F: Vessels in each field were counted and plotted ±SEM (n ≥10 fields per group; *P < 0.00003 when comparing vessel number from line 22 and control line 6A8).

Article Snippet: Anti-human HRGP monoclonal antibody (MAB1869) was purchased from R&D Systems (Minneapolis, MN) and anti-human N-terminal BAI1 polyclonal antibody was previously described.

Techniques: Expressing, Clone Assay, Stable Transfection, Injection, Control, Staining, Avidin-Biotin Assay

Journal:

Article Title: Histidine-Rich Glycoprotein Modulates the Anti-Angiogenic Effects of Vasculostatin

doi: 10.2353/ajpath.2010.090782

Figure Lengend Snippet: HRGP increases tumor growth in Vstat120-expressing glioma cells transplanted orthotopically into brains of nude mice. Cells from five LN229 cloned glioma cell lines (parental LN229, line 6A8 expressing Vstat120 alone, and lines expressing both Vstat120 and HRGP; lines 5, 22, and 23) were implanted into the brains of immunocompromised mice (n = 6 mice per group). A: Cells not used in the implantations were plated and tested for HRGP expression via Western blot. B: Quantification of the tumor area for each implanted group. Each section with the largest tumor area within 1 mm anterior to posterior of the injection point was used for analysis. To quantify, the area of tumor was calculated within the normal shape of the brain and any tumor area that expanded beyond that was not included. The percent tumor area represents the ratio of the measured tumor area/the area of the total hemisphere × 100%. Error bars are expressed as ±SEM. Significance was assessed by Student t test; *P < 0.03, **P < 0.003; n.s indicates not significant. C–I: HRGP increases vessel size and number in Vstat120 orthotopically implanted gliomas. Tumor sections were stained with anti-vWF and goat anti-rabbit alexa fluor 488 to visualize tumor vessels. Pictures were taken under ×40 magnification. The peripheral regions of each tumor that were in contact with the nontumorous tissue were used to calculate vessel density. Parental LN229 glioma line showed extensive blood vessel staining (green) within the tumor (cells stained blue with DAPI), especially at the tumor-brain tissue interface (C) with an average number of 19.4 per field (I – LN229 – lane 5). LN229 cells that stably express Vstat120 (control line 6A8 – representative images of two tumors are shown in D and E) showed dramatically reduced vessel number and size (white arrows). Vessel number per field was reduced to 2.8 in the presence of Vstat120 (I – 6A8 – lane 4). Tumors with both Vstat120 and HRGP constitutively expressed regain a significant amount of vascularity when compared with tumors with Vstat120 alone (F–H) with and average vessel density of 11, 9.9, and 11.8 per field (I – 5 – lane 1, 22 – lane 2 and 23 – lane 3) respectively. *P < 0.0001 when comparing the HRGP lines with the control 6A8 line.

Article Snippet: Anti-human HRGP monoclonal antibody (MAB1869) was purchased from R&D Systems (Minneapolis, MN) and anti-human N-terminal BAI1 polyclonal antibody was previously described.

Techniques: Expressing, Clone Assay, Western Blot, Injection, Staining, Stable Transfection, Control

Journal:

Article Title: Histidine-Rich Glycoprotein Modulates the Anti-Angiogenic Effects of Vasculostatin

doi: 10.2353/ajpath.2010.090782

Figure Lengend Snippet: Formalin-fixed and paraffin-embedded biopsy sections of glioblastoma tumor (A and C) and of normal brain tissue removed during epilepsy surgery (B and D) were deparaffinized, blocked, and reacted with 0.7 μg/ml anti-HRGP antibody (A and B) or control IgG (C and D). This was followed by a biotin-conjugated secondary antibody, reaction with strepavidin, the DAB substrate, and hematoxylin counterstaining. Slides were viewed and photographed in a Leica DMR microscope at ×40 magnification. Arrows denote the basement membrane of blood vessels, and the arrowheads in panel A denote endothelial cells that are not stained with the anti-HRGP antibody. The tumor cells in the GBM biopsy are negative for HRGP staining.

Article Snippet: Anti-human HRGP monoclonal antibody (MAB1869) was purchased from R&D Systems (Minneapolis, MN) and anti-human N-terminal BAI1 polyclonal antibody was previously described.

Techniques: Control, Microscopy, Membrane, Staining

Journal:

Article Title: Histidine-Rich Glycoprotein Modulates the Anti-Angiogenic Effects of Vasculostatin

doi: 10.2353/ajpath.2010.090782

Figure Lengend Snippet: Expression of HRGP in Glioblastoma Tumor and Normal Brain

Article Snippet: Anti-human HRGP monoclonal antibody (MAB1869) was purchased from R&D Systems (Minneapolis, MN) and anti-human N-terminal BAI1 polyclonal antibody was previously described.

Techniques: Expressing

Journal:

Article Title: Histidine-Rich Glycoprotein Modulates the Anti-Angiogenic Effects of Vasculostatin

doi: 10.2353/ajpath.2010.090782

Figure Lengend Snippet: A and B: Model of the effect of Vstat120 on endothelial cells and the modulating role of HRGP. A: In the microenvironment of a brain tumor, growth factors such as basic fibroblast growth factor or vascular endothelial growth factor are secreted and induce angiogenesis via interaction with specific receptors on the vascular endothelium. At the same time, the extracellular domain of BAI1 is cleaved from the glial cell membrane liberating Vstat120 (V), which then interacts with the endothelial cell receptor CD36. Downstream signaling induced by CD36 through src kinases fyn and lyn and p38 MAP kinase diverts the pro-angiogenic growth factor signals to caspase-mediated apoptotic signals. B: As a consequence of vascular inflammation and/or increased permeability of the tumor neovessels induced by VEGF, plasma or platelet HRGP is deposited in the tumor bed where it acts as a decoy receptor for Vstat120. Vstat120 is then unable to bind its cell surface receptors and endothelial cell proliferation, migration, and tube formation occur, leading to increased vessel density and tumor growth.

Article Snippet: Anti-human HRGP monoclonal antibody (MAB1869) was purchased from R&D Systems (Minneapolis, MN) and anti-human N-terminal BAI1 polyclonal antibody was previously described.

Techniques: Membrane, Permeability, Clinical Proteomics, Migration