Journal: The Journal of infectious diseases

Article Title: Osteopontin impairs host defense during pneumococcal pneumonia.

doi: 10.1093/infdis/jir185

Figure Lengend Snippet: Figure 1. Pulmonary and plasma osteopontin concentrations are elevated during pneumococcal pneumonia. Osteopontin concentrations in (A) lung and (B) plasma before and 6, 24, and 48 h after infection with 104 colony-forming units of Streptococcus pneumoniae. Data are expressed as mean 6 standard error of the mean (SEM); n 5 8 mice per group. Asterisk, P , .05; double asterisk, P , .01; triple asterisk, P , .001, compared with t 5 0. Osteopontin concentrations in culture supernatants after incubation of (C) MH-S cells and (D) primary alveolar macrophages with medium or growth- arrested S. pneumoniae (multiplicity of infection, 1:6 and 1:60 for MH-S cells; 1:20 and 1:200 for primary alveolar macrophages) for 4 h (MH-S cells) or 20 h (primary alveolar macrophages). Data are expressed as mean 6 SEM; n 5 3 per group. Asterisk, P ,.05, compared with medium. OPN, osteopontin.

Article Snippet: Effect of Osteopontin on S. pneumoniae Viability, Phagocytosis, and Phagolysosomal Fusion S. pneumoniae or Staphylococcus (S.) aureus (Newman strain) (1 3 106 bacteria/mL) was incubated in sterile normal saline in the presence of 0.8–800 ng/mL recombinant mouse osteopontin (rOPN;,1.0 endotoxin units per 1 lg as determined by the LAL assay; R&D Systems), 800 ng/mL boiled rOPN (30 min at 100 C), 800 ng/mL bovine serum albumin (BSA), or normal saline only at 37 C for 6 h. At indicated time points the number of bacteria was determined.

Techniques: Clinical Proteomics, Infection, Incubation

Journal: The Journal of infectious diseases

Article Title: Osteopontin impairs host defense during pneumococcal pneumonia.

doi: 10.1093/infdis/jir185

Figure Lengend Snippet: Figure 2. Prolonged survival and reduced bacterial growth in osteopontin knockout (KO) mice. A, Percentage survival of wild-type (WT) mice (filled symbols) and osteopontin KO mice (open symbols) after intranasal infection with 104 colony-forming units (CFU) of Streptococcus pneumoniae (n 5 14 mice per group). P value indicates the difference between groups. WT (gray) and osteopontin KO (white) mice were infected with 104 CFU of S. pneumoniae, and bacterial loads were determined 6, 24, and 48 h after infection in (B) lung, (C) blood, and (D) spleen. Data are expressed as box-and- whisker diagrams depicting the smallest observation, lower quartile, median, upper quartile, and largest observation; n 5 8 mice per group; asterisk, P ,.05; double asterisk, P ,.01; triple asterisk, P ,.001, compared with WT mice. Note to panels C and D: none of the mice in either group displayed positive blood or spleen culture results 6 h after infection; at 24 h, S. pneumoniae could be cultured from samples of the blood of only 3 of 8 osteopontin KO mice, compared with 7 of 8 WT mice and from the spleen tissue of only 1 of 7 osteopontin KO mice, compared with 7 of 8 WT mice (P , .05 and P , .01, respectively). OPN, osteopontin.

Article Snippet: Effect of Osteopontin on S. pneumoniae Viability, Phagocytosis, and Phagolysosomal Fusion S. pneumoniae or Staphylococcus (S.) aureus (Newman strain) (1 3 106 bacteria/mL) was incubated in sterile normal saline in the presence of 0.8–800 ng/mL recombinant mouse osteopontin (rOPN;,1.0 endotoxin units per 1 lg as determined by the LAL assay; R&D Systems), 800 ng/mL boiled rOPN (30 min at 100 C), 800 ng/mL bovine serum albumin (BSA), or normal saline only at 37 C for 6 h. At indicated time points the number of bacteria was determined.

Techniques: Knock-Out, Infection, Whisker Assay, Cell Culture

Journal: The Journal of infectious diseases

Article Title: Osteopontin impairs host defense during pneumococcal pneumonia.

doi: 10.1093/infdis/jir185

Figure Lengend Snippet: Figure 3. Decreased lung histopathology in osteopontin knockout (KO) mice. Representative lung histology of wild-type (WT) (A, D, G) and osteopontin KO (B, E, H ) mice at 6 h (A–C ), 24 h (D–F ), and 48 h (G–I) after intranasal infection with 104 CFU of Streptococcus pneumoniae. The lung sections are representative for 8 mice per group per time point. Hematoxilin and eosin staining, original magnification, 310. Inflammation scores are expressed as mean 6 standard error of the mean (WT mice, black bars; osteopontin KO mice, white bars; n 5 8 mice per group). Double asterisk, P ,.01, compared with WT mice. OPN, osteopontin.

Article Snippet: Effect of Osteopontin on S. pneumoniae Viability, Phagocytosis, and Phagolysosomal Fusion S. pneumoniae or Staphylococcus (S.) aureus (Newman strain) (1 3 106 bacteria/mL) was incubated in sterile normal saline in the presence of 0.8–800 ng/mL recombinant mouse osteopontin (rOPN;,1.0 endotoxin units per 1 lg as determined by the LAL assay; R&D Systems), 800 ng/mL boiled rOPN (30 min at 100 C), 800 ng/mL bovine serum albumin (BSA), or normal saline only at 37 C for 6 h. At indicated time points the number of bacteria was determined.

Techniques: Histopathology, Knock-Out, Infection, Staining

Journal: The Journal of infectious diseases

Article Title: Osteopontin impairs host defense during pneumococcal pneumonia.

doi: 10.1093/infdis/jir185

Figure Lengend Snippet: Figure 4. Osteopontin stabilizes Streptococcus pneumoniae viability in vitro. A, S. pneumoniae in saline (106 colony-forming units [CFU]/mL) was incubated with increasing doses (0.8–800 ng/mL) of recombinant osteopontin (black symbols) or saline (white symbols), and the viability of S. pneumoniae was determined over 6 h at 37C. B, S. pneumoniae in saline (106 CFU/mL) was incubated with 800 ng/mL recombinant osteopontin (filled squares), 800 ng/mL boiled recombinant osteopontin (open squares), 800 ng/mL bovine serum albumin (triangles), or saline (circles), and the viability of S. pneumoniae was determined over 6 h at 37C. Dashed lines depict detection limits. C, Osteopontin binds to S. pneumoniae. Enzyme-linked immunosorbent assay plates were coated or not coated with 1 3 108 CFU/mL S. pneumoniae type 3 (ATCC 6303) or serotype 2 (D39); coating with anti-osteopontin IgG was used as positive control. Binding was assessed using biotin-labeled recombinant mouse osteopontin. Data are means 6 standard error (n 5 4–6). Double asterisk, P , .01 vs buffer. OPN, osteopontin.

Article Snippet: Effect of Osteopontin on S. pneumoniae Viability, Phagocytosis, and Phagolysosomal Fusion S. pneumoniae or Staphylococcus (S.) aureus (Newman strain) (1 3 106 bacteria/mL) was incubated in sterile normal saline in the presence of 0.8–800 ng/mL recombinant mouse osteopontin (rOPN;,1.0 endotoxin units per 1 lg as determined by the LAL assay; R&D Systems), 800 ng/mL boiled rOPN (30 min at 100 C), 800 ng/mL bovine serum albumin (BSA), or normal saline only at 37 C for 6 h. At indicated time points the number of bacteria was determined.

Techniques: In Vitro, Saline, Incubation, Recombinant, Enzyme-linked Immunosorbent Assay, Positive Control, Binding Assay, Labeling

Journal: The Journal of infectious diseases

Article Title: Osteopontin impairs host defense during pneumococcal pneumonia.

doi: 10.1093/infdis/jir185

Figure Lengend Snippet: Figure 5. Similar bacterial growth during pneumococcal sepsis. Bacterial loads in (A) blood, (B) lung, (C) liver, and (D) spleen from wild-type (WT; gray) and osteopontin knockout (OPN KO; white) mice at 24 and 48 h after intravenous injection with 105 colony-forming units (CFU) of Streptococcus pneumoniae. Data are expressed as box-and-whisker diagrams depicting the smallest observation, lower quartile, median, upper quartile, and largest observation; n 5 8 mice per group. Dashed line depicts detection limit.

Article Snippet: Effect of Osteopontin on S. pneumoniae Viability, Phagocytosis, and Phagolysosomal Fusion S. pneumoniae or Staphylococcus (S.) aureus (Newman strain) (1 3 106 bacteria/mL) was incubated in sterile normal saline in the presence of 0.8–800 ng/mL recombinant mouse osteopontin (rOPN;,1.0 endotoxin units per 1 lg as determined by the LAL assay; R&D Systems), 800 ng/mL boiled rOPN (30 min at 100 C), 800 ng/mL bovine serum albumin (BSA), or normal saline only at 37 C for 6 h. At indicated time points the number of bacteria was determined.

Techniques: Knock-Out, Injection, Whisker Assay

Journal: The Prostate

Article Title: Tranilast inhibits hormone refractory prostate cancer cell proliferation and suppresses transforming growth factor beta1-associated osteoblastic changes.

doi: 10.1002/pros.20975

Figure Lengend Snippet: Fig. 4. A:ThechangesinTGF-b1secretioninprostatecancercelllines,SaOS-2,andbone-derivedstromalcellsbytranilastwereexaminedby ELISA as describedin Materials and Methods Section.Bars, SD.B: Osteopontin (1mg/ml) was addeddaily and 300mmol/L tranilastwas added everyotherdaytoLNCaP-SFcells.Woundphotographsweretakenunderphase-contrastmicroscopyatinitialtime(0hr)andtheterminationof theexperiments(96hr).C:Resultsarepresentedas thepercentagetocontrolin5%CCS(*P < 0.05,**P < 0.01,***P < 0.001).

Article Snippet: Cells were treated with recombinant human osteopontin (R&D Systems) alone or with tranilast in 5% CCS or serum free medium.

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Journal of the American Heart Association

Article Title: Defining the Cardiac Fibroblast Secretome in a Fibrotic Microenvironment

doi: 10.1161/jaha.120.017025

Figure Lengend Snippet: Figure 5. Changes in protein secretion in conditioned media match changes in gene expression in both in vitro and in vivo models of fibrosis. A, Schematic of experimental layout for measuring changes in protein secretion in conditioned media and gene expression in cardiac fibroblasts (CFs) cultured on hydrogels. B, Protein changes in select cytokines from cytokine array (normalized to 6 kPa condition, N=3, error bars show mean and SEM, 1-way ANOVA, *P≤0.05, **P≤0.01). C, Schematic representation of experimental design to compare changes in cytokine gene expression in vitro with changes in vivo with isoproterenol (ISO). D, Changes in gene expression of cytokines from CFs cultured on hydrogels±TGFβ1 (transforming growth factor β 1) for 5 days (N=3, error bars show mean and SEM, 1-way ANOVA, *P≤0.05, **P≤0.01, ***P≤0.001, ****P≤0.0001). E, Changes in gene expression of cytokines from CFs isolated from rats treated with vehicle or ISO (4 mg/kg per day for 3 days, N=6 for each group, error bars show mean and SEM, 2-tailed unpaired T test, *P≤0.05, **P≤0.01, ****P≤0.0001). FC indicates fold change; IGFBP, insulin growth factor binding protein; OPG, osteoprotegerin; OPN, osteopontin; VEGF, vascular endothelial growth factor; and WISP-1, Wnt1 inducible signaling pathway protein 1.

Article Snippet: IGF-1 (R&D Systems 4326-RG-050) and osteopontin (R&D Systems 6359-OP-050) were added NRVMs in serum-free media and cultured for 48 hours before NRVMs were trypsinized and cell size was measured by Coulter Counter.

Techniques: Gene Expression, In Vitro, In Vivo, Cell Culture, Isolation, Binding Assay

Journal: Journal of the American Heart Association

Article Title: Defining the Cardiac Fibroblast Secretome in a Fibrotic Microenvironment

doi: 10.1161/jaha.120.017025

Figure Lengend Snippet: Figure 6. The cardiac fibroblast (CF) secretome increases neonatal rat ventricular myocyte (NRVM) size. A, Schematic of conditioned medium (CM) treatment of NRVMs (48 hours treatment with conditioned media from CFs). B, Coulter counter measurements of NRVMs treated for 48 hours with CM from CFs with or without 3 μmol/L SD208 to inhibit effects caused by TGFβ1 (transforming growth factor β 1) (N=CM from 3 CF isolations, 4 rats pooled per isolation, error bars show mean and SEM, 1-way ANOVA, *P≤0.05, **P≤0.01). C, Coulter counter measurements of NRVM volume when treated with different concentrations of osteopontin (OPN), insulin growth factor 1 (IGF-1), or phenylephrine (PE) (10 μmol/L), (N=2 isolations of NRVMs, N=3 for controls [same controls used for OPN and IGF-1 treatment], error bars show SD, 1-way ANOVA, significance compared with untreated, *P≤0.05, **P≤0.01). D, Schematic of CF signaling to NRVMs to induce hypertrophy through IGF-1 signaling.

Article Snippet: IGF-1 (R&D Systems 4326-RG-050) and osteopontin (R&D Systems 6359-OP-050) were added NRVMs in serum-free media and cultured for 48 hours before NRVMs were trypsinized and cell size was measured by Coulter Counter.

Techniques: Isolation

Journal: Journal of the American Heart Association

Article Title: Defining the Cardiac Fibroblast Secretome in a Fibrotic Microenvironment

doi: 10.1161/jaha.120.017025

Figure Lengend Snippet: Figure 7. Schematic of TGFβ (transforming growth factor β) signaling and signaling through extracellular matrix (ECM) interactions to influence cytokine secretion. Cytokines that changed with TGFβ signaling vs ECM stiffness sensing are listed, and insulin growth factor 1 (IGF-1), secreted by TGFβ-activated myofibroblasts, caused hypertrophy. GM-CSF indicates granulocyte-macrophage colony-stimulating factor; IGFBP, insulin growth factor binding protein; OPN, osteopontin; OPG, osteoprotegerin; WISP-1, Wnt1 inducible signaling pathway protein 1; and CCL, Chemokine (C-C motif) ligand.

Article Snippet: IGF-1 (R&D Systems 4326-RG-050) and osteopontin (R&D Systems 6359-OP-050) were added NRVMs in serum-free media and cultured for 48 hours before NRVMs were trypsinized and cell size was measured by Coulter Counter.

Techniques: Binding Assay

Journal: PLoS ONE

Article Title: Osteopontin Enhances the Expression and Activity of MMP-2 via the SDF-1/CXCR4 Axis in Hepatocellular Carcinoma Cell Lines

doi: 10.1371/journal.pone.0023831

Figure Lengend Snippet: The invasion assay was set up in transwell chambers. Cell culture inserts with 8.0-µm pore diameter were used to separate the top and bottom chambers. 60 µl of ECM gel solution was added to the upper compartment of each cell culture insert and dried overnight under laminar air flow. SMMC7721 parent cells, vector controls, and CXCR4 miRNA clones (1-4, 2-4) were plated onto the membrane of the top chamber, and rhOPN was administered to the bottom chamber. After 48 hours, the cells that had invaded to the lower surface of the membrane were enumerated. ( A ) Bright-field image of cells migrated to the bottom of chambers on the inserts (200× original magnification). ( B ) Quantification of cell invasion. The open bars represent no osteopontin, the filled bars represent rhOPN treatment. In each chamber, six fields were counted at 200× magnification for each condition by two investigators. * indicates P <0.05 versus control. The data are representative of three experiments.

Article Snippet: rhOPN (Recombinant human Osteopontin/his) (#1433-OP/CF) was purchased from R&D Systems (USA).

Techniques: Invasion Assay, Cell Culture, Plasmid Preparation, Clone Assay

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Osteopontin Isoforms Differentially Promote Arteriogenesis in Response to Ischemia via Macrophage Accumulation and Survival

doi: 10.1038/s41374-018-0094-8

Figure Lengend Snippet: A. Illustration of OPN isoform primary domain structure. Each block corresponds to an exon (numbered). OPNa is full length (top), OPNb lacks exon 5 (middle), and OPNc lacks exon 4 (bottom). Expanded amino acid sequences of exons 4 and 5, absent in OPNc and OPNb, respectively, are included. B. OPN primers were used to measure OPNa (277bp), OPNb (235bp), and OPNc (196bp) mRNA levels in tissue samples from non-ischemic tissues and tissues from PAD patients with critical limb ischemia by RT-PCR (n=3–5). Isoform plasmid DNA controls and beta actin for loading are both shown. C. To investigate if OPN isoforms differentially affect collateral vessel formation in vivo , the hindlimb ischemia was performed on the following groups: WT, OPN −/− , or OPN −/− mice treated (trx) with lentivirus (LV) to overexpress GFP, OPNa, OPNb, or OPNc. Perfusion was measured by Laser Doppler perfusion imaging (LDPI). Representative LDPI images 14 days post-HLI are shown. D. Ischemic limb (IL) perfusion was quantified and normalized to the contralateral non-ischemic limb (NIL) and compared across groups. * p<0.05, † p<0.001 vs. GFP; d14, n=6. E. To determine if the OPN isoform effects on perfusion translate to increased functional limb use, animals were given free access to a running wheel (d7 post-HLI) and allowed to run for 7 days. Total distance run (meters) by was plotted for all groups as a measure of limb function. *p<0.05, † p<0.001 vs. GFP; n=9–10.

Article Snippet: For macrophage polarization studies, 3 hours after plating cells were stimulated with 10% FBS-RPMI with 100 ng/mL purified recombinant human OPNa, OPNb, or OPNc (cat# TP304803, TP305118, and TP31059; Origene, Rockville, MD), or were stimulated with 20 ng/mL of either interferon gamma (INFγ) or interleukin (IL)-4 for 48 hours (cat# 554587 and 550067; BD Biosciences) as positive controls for macrophage polarization.

Techniques: Blocking Assay, Reverse Transcription Polymerase Chain Reaction, Plasmid Preparation, In Vivo, Imaging, Functional Assay

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Osteopontin Isoforms Differentially Promote Arteriogenesis in Response to Ischemia via Macrophage Accumulation and Survival

doi: 10.1038/s41374-018-0094-8

Figure Lengend Snippet: To determine if OPN isoforms differentially affect arteriogenesis, tissue sections from animals 14 days post-HLI treated (trx) with lentivirus (LV) to overexpress OPN isoform a, b, or c were stained with α smooth muscle actin (α-SMA). α-SMA positive vessel numbers and sizes were quantified as a readout for arteriogenesis. A. The number of α-SMA positive vessels was counted across treatment groups and plotted (p = ns). B. α-SMA positive vessel sizes were measured in WT or OPN −/− mice treated (trx) with lentivirus (LV) to overexpress GFP, OPNa, OPNb, or OPNc. The number of vessels measured within the arteriole (10 – 200 μm 2 ), small artery (200 – 700 μm 2 ) and large artery (1000 – 2500 μm 2 ) size ranges were compared across all animal groups. Data are expressed as % change compared to +LV-GFP (control). *p<0.05 vs. OPNa, † p<0.001 vs. OPNb; n=8–10. C. Representative histology images from 14 days post-HLI stained with α-SMA are shown. α-SMA stain is red and counterstain is violet. Scale bars = 500 μm.

Article Snippet: For macrophage polarization studies, 3 hours after plating cells were stimulated with 10% FBS-RPMI with 100 ng/mL purified recombinant human OPNa, OPNb, or OPNc (cat# TP304803, TP305118, and TP31059; Origene, Rockville, MD), or were stimulated with 20 ng/mL of either interferon gamma (INFγ) or interleukin (IL)-4 for 48 hours (cat# 554587 and 550067; BD Biosciences) as positive controls for macrophage polarization.

Techniques: Staining