Journal: Journal of molecular medicine (Berlin, Germany)

Article Title: Host and Pathogen Hyaluronan Signal Through Human Siglec-9 to Suppress Neutrophil Activation

doi: 10.1007/s00109-015-1341-8

Figure Lengend Snippet: Specific recognition of hyaluronan by the Ig-like-V-set domain of hSiglec-9. (a) Immobilized high molecular weight-hyaluronan (HMW-HA) was probed with human Siglec–Fc and CD44–Fc chimeras and binding evaluated by using an anti-human IgG-HRP. (b) Binding of hSiglec-9–Fc to immobilized HMW-HA was performed in the presence of increasing concentrations of HMW-HA, heparan sulfate, chondroitin sulfate or heparin. (c, d) To map the hSiglec-9 domain responsible for HA recognition, binding of hSiglec-9–Fc to immobilized HMW-HA was compared to binding of hSiglec-9–Fc with an Arg→Ala mutation in the V-set domain (hSiglec-9R120K), a fusion protein construct of the hSiglec-9 V-set domain + the second Ig-like domain (C2-set) of hSiglec-7 + human IgG Fc tail (hSiglec-9V-7C2–Fc), a fusion protein construct encompassing V-set domain of hSiglec-7 + C2-set of hSiglec-9 + human IgG Fc tail (Siglec-7V-9C2–Fc), a fusion protein construct of the hSiglec-9 V-set domain + first C2-set domain + human IgG Fc tail (Siglec-9–Fc 2D). Results are expressed as the mean ± SD. All experiments were performed in triplicate, repeated 3 times (a, d) or 2 times (b). One-way ANOVA with Dunnett’s multiple comparison test; P < 0.001 (***).

Article Snippet: Siglec-Fc proteins were purified from culture supernatant by adsorption to protein A-Sepharose (GE Healthcare); human CD44–Fc chimera was purchased from R&D Systems. hSiglecs-Fc and CD44–Fc were treated with 25 mU and 5 mU of Arthrobacter ureafaciens sialidase (AUS) for 60 min to remove Sias, prior to elution from the protein A-Sepharose.

Techniques: Molecular Weight, Binding Assay, Mutagenesis, Construct, Comparison

Journal: Journal of molecular medicine (Berlin, Germany)

Article Title: Host and Pathogen Hyaluronan Signal Through Human Siglec-9 to Suppress Neutrophil Activation

doi: 10.1007/s00109-015-1341-8

Figure Lengend Snippet: High molecular weight hyaluronan (HMW-HA) is recognized by hSiglec-9 on human neutrophils. (a) To evaluate the molecular size of hyaluronan (HA) responsible for binding to hSiglec-9, high molecular weigh HA (HMW-HA; >1,000 kDa), low molecular weigh (200 – 300 kDa-HA and 25 – 75 kDa-HA), nano-HA (nonamers) and oligo-HA (hexamers) fragments were added to compete with binding of hSiglec-9–Fc to immobilized HMW-HA plates. Binding was evaluated using an anti-human IgG-HRP. Experiment performed in triplicate and repeated 3 times; results are expressed as mean ± SD. (b) Flow cytometry reveals constitutive expression of Siglec-9 and CD44 on human neutrophils from 9 different donors; geometric mean ± 95% confidence interval. (c) Binding of FITC-labeled HMW-HA to neutrophils of these donors was evaluated by flow cytometry; geometric mean ± 95% confidence interval. (d) Human neutrophils were pretreated with anti-Siglec-9 monoclonal antibodies, anti-human CDw329 (BD Pharmingen, #550906) and anti-human Siglec-9 (R&D Systems, #BAF1139) and effects on binding to FITC-labeled HMW-HA determined. CDw329 Ab blocked binding of HMW-HA, but not GBS capsule (Fig. 5b) and was designated “α-Sig-9(HA)”; in contrast, the R&D Systems Ab blocked recognition of GBS capsule (Fig. 5b), but did not interfere with binding to HMW-HA, and was thus designated “α-Sig-9(Sia)”. Data pooled from five independent experiments in triplicate; data represent the mean ± SD. (e, f) Calcein-labeled human neutrophils were pretreated with α-Sig-9(HA), α-Sig-9(Sia) and α-CD44 mAbs, added to wells coated with immobilized HMW-HA to facilitate adherence, then unbound neutrophils washed away. Remaining neutrophils were lifted and enumerated. Experiment performed in triplicate and repeated 5 times; results are expressed as mean ± SD. One-way ANOVA with Dunnett’s multiple comparison test; P < 0.001 (***).

Article Snippet: Siglec-Fc proteins were purified from culture supernatant by adsorption to protein A-Sepharose (GE Healthcare); human CD44–Fc chimera was purchased from R&D Systems. hSiglecs-Fc and CD44–Fc were treated with 25 mU and 5 mU of Arthrobacter ureafaciens sialidase (AUS) for 60 min to remove Sias, prior to elution from the protein A-Sepharose.

Techniques: Molecular Weight, Binding Assay, Flow Cytometry, Expressing, Labeling, Comparison

Journal: Journal of molecular medicine (Berlin, Germany)

Article Title: Host and Pathogen Hyaluronan Signal Through Human Siglec-9 to Suppress Neutrophil Activation

doi: 10.1007/s00109-015-1341-8

Figure Lengend Snippet: Group A Streptococcus (GAS) engages hSiglec-9 via its surface hyaluronan capsule. (a) Human Siglec-9–Fc was immobilized to ELISA wells using protein A, and binding of FITC-labeled forms of WT GAS, its isogenic HA capsule-deficient mutant (ΔhasA), an animal-passaged hyperencapsulated (AP) derivative and its isogenic HA capsule-deficient mutant (AP ΔhasA) were evaluated; results are expressed as mean ± SD and repeated 5 times in triplicate with similar results; representative experiment shown. One-Way ANOVA with Dunnett’s multiple comparison test; P < 0.001 (***) (b) Human Siglec-9–Fc chimera was immobilized to ELISA wells via protein A in the presence of α-Sig-9(HA), α-Sig-9(Sia) or isotype control Abs and binding of FITC-labeled WT GAS or sialic acid-expressing serotype III group B Streptococcus (GBS) evaluated. (c) Human Siglec-9/9R120K/5/6/7/11 and human CD44-Fc chimera were immobilized to ELISA wells via protein A. Binding of FITC-labeled WT GAS was evaluated. Results represent mean ± SD; triplicate wells, representative experiment depicted of 5 independent repeats with similar results, performed in triplicate. (d) hSiglec-9–Fc was immobilized to ELISA plates using protein A, then wells were pretreated with HMW-HA, HMW-heparan sulfate or heparin over the indicated range of concentrations. Binding of FITC-labeled WT GAS was evaluated.

Article Snippet: Siglec-Fc proteins were purified from culture supernatant by adsorption to protein A-Sepharose (GE Healthcare); human CD44–Fc chimera was purchased from R&D Systems. hSiglecs-Fc and CD44–Fc were treated with 25 mU and 5 mU of Arthrobacter ureafaciens sialidase (AUS) for 60 min to remove Sias, prior to elution from the protein A-Sepharose.

Techniques: Enzyme-linked Immunosorbent Assay, Binding Assay, Labeling, Mutagenesis, Comparison, Expressing

Journal: Journal of molecular medicine (Berlin, Germany)

Article Title: Host and Pathogen Hyaluronan Signal Through Human Siglec-9 to Suppress Neutrophil Activation

doi: 10.1007/s00109-015-1341-8

Figure Lengend Snippet: HMW-HA binding to hSiglec-9 induces SHP-1 recruitment and blunts neutrophil NET production and oxidative burst. (a) 1.2 × 107 human neutrophils were incubated for 30 min + 10 µg/ml of high molecular weight hyaluronan (HMW-HA) + 25 nM PMA. Cell lysates were immunoprecipitated with α-Siglec9 and SHP-1 recruitment was visualized by western blot analysis; results were repeated 2 times with similar results; representative experiment with relative densitometry values is shown. (b) Neutrophils were pretreated with α-Sig-9(HA), α-Sig-9(Sia) or α-CD44 Abs, incubated with 10 µg/ml of HMW-HA and activated for 30 min with PMA. Reactive oxygen species (ROS) release was measured with the OxyBURST Green H2HFF BSA probe and results expressed as mean fluorescence intensity (MFI) ± SD; experiment repeated 5 times with similar results; representative experiment is shown. (c) Neutrophils were pretreated α-Sig-9(HA), α-Sig-9(Sia), or α-CD44 mAbs, then incubated with 10 µg/ml of HMW-HA and activated with PMA for 3 h; production of neutrophil extracellular traps (NETs) visualized by staining for DAPI (DNA, blue) + anti-myeloperoxidase/AlexaFluor488 (green); representative fields at 20x magnification is shown; experiment performed in triplicate and repeated 5 times. (d) NET production was quantified by Quant-iT™ PicoGreen® assay for extracellular DNA; results are expressed as mean ± SD; experiment repeated 3 times in triplicate with similar results; representative experiment shown. One-way ANOVA with Dunnett’s multiple comparison test; P < 0.001 (***).

Article Snippet: Siglec-Fc proteins were purified from culture supernatant by adsorption to protein A-Sepharose (GE Healthcare); human CD44–Fc chimera was purchased from R&D Systems. hSiglecs-Fc and CD44–Fc were treated with 25 mU and 5 mU of Arthrobacter ureafaciens sialidase (AUS) for 60 min to remove Sias, prior to elution from the protein A-Sepharose.

Techniques: Binding Assay, Incubation, Molecular Weight, Immunoprecipitation, Western Blot, Fluorescence, Staining, Picogreen Assay, Comparison

Journal: Journal of molecular medicine (Berlin, Germany)

Article Title: Host and Pathogen Hyaluronan Signal Through Human Siglec-9 to Suppress Neutrophil Activation

doi: 10.1007/s00109-015-1341-8

Figure Lengend Snippet: Group A Streptococcus (GAS) binding to hSiglec-9 via its surface HMW-HA capsule blunts neutrophil oxidative burst, NET responses and bactericidal activity. (a, b) Neutrophils were labeled with OxyBURST Green H2HFF BSA in the presence of α-Sig-9(HA), α-Sig-9(Sia) or α-CD44 mAbs, infected with WT or isogenic ΔhasA GAS at MOI = 20 for 30 min and oxidative burst measured by FACS; results are expressed as MFI ±SD and repeated twice with similar results; representative experiment is shown. (c) PMA-stimulated neutrophils (5 × 105 cells) were pretreated with α-Sig-9(HA), α-Sig-9(Sia), or α-CD44 mAbs and exposed for 3 h to MOI = 10 of GAS (DNase mutant) that had been pretreated or not with hyaluronidase to remove HA capsule and NET production visualized by staining for DAPI (DNA, blue) + anti-myeloperoxidase/AlexaFluor488 (green); results are repeated 5 times in triplicate, representative fields at 32 × magnification is shown. (d) NET production was quantified by Quant-iT™ PicoGreen® assay for extracellular DNA; results are expressed as mean ± SD and repeated 2 times with similar results; representative experiment shown. (e) Neutrophils were pretreated with α-Sig-9(HA), α-Sig-9(Sia) or α-CD44 mAbs, infected with WT or isogenic ΔhasA GAS at multiplicity of infection (MOI) = 10 for 30 min, then cells lysed and dilutions plated on agar for enumeration of colony forming units to evaluate neutrophil killing of GAS. Data represent the mean + SD of triplicates; repeated 4 times with similar results; representative experiment shown. One-Way ANOVA with Dunnett’s multiple comparison test; P < 0.001 (***) or P < 0.05 (*).

Article Snippet: Siglec-Fc proteins were purified from culture supernatant by adsorption to protein A-Sepharose (GE Healthcare); human CD44–Fc chimera was purchased from R&D Systems. hSiglecs-Fc and CD44–Fc were treated with 25 mU and 5 mU of Arthrobacter ureafaciens sialidase (AUS) for 60 min to remove Sias, prior to elution from the protein A-Sepharose.

Techniques: Binding Assay, Activity Assay, Labeling, Infection, Mutagenesis, Staining, Picogreen Assay, Comparison

Journal: bioRxiv

Article Title: CD44 Controls Endothelial Proliferation and Functions as Endogenous Inhibitor of Angiogenesis

doi: 10.1101/049494

Figure Lengend Snippet: Increased angiogenesis in CD44-null mice. ( A ) Angiogenesis was analysed in C3H, C57BL/6, and in wild-type and Cd44 −/− mice of mixed genetic backgrounds (WT mix and Cd44KO mix, respectively). Basement membrane extract–filled angioreactors containing premixed FGF-2, VEGF and heparin or PBS for uninduced controls were implanted SC into the flanks of mice. Each mouse received 2 angioreactors, 1 per flank. 14 days after implantation, angioreactors were resected and the population of ECs within the angioreactor matrix was assessed by FITC-lectin staining. The number of fluorescent cells was quantitated by microplate reader. Raw readings from independent experiments were scaled by dividing by their quadratic mean. N = 2–5 mice per condition from 2 independent experiments. P value is from Student’s t-test. ( B ) Angiogenesis in Cd44 −/− mice and their heterozygous ( Cd44 +/− ) and wild-type ( Cd44 +/+ ) littermate that had been backcrossed six generations to the C57BL/6 background. The data are represented as the mean ± SEM. Each dot represents the mean of two angioreactors for an individual mouse. N = 8 mice per condition from 2 independent experiments. P values are from ANOVA post hoc comparisons using the Tukey HSD test.

Article Snippet: After blocking, samples and standards (Recombinant Mouse CD44 Fc Chimera; R&D Systems) were incubated in wells for 30 min at 37°C.

Techniques: Membrane, Staining

Journal: bioRxiv

Article Title: CD44 Controls Endothelial Proliferation and Functions as Endogenous Inhibitor of Angiogenesis

doi: 10.1101/049494

Figure Lengend Snippet: Recombinant CD44-3MUT Fc fusion protein inhibits angiogenesis in vivo . First, the assay and quantitation were performed similarly to those described in , except that each mouse received 4 angioreactors, 2 per flank. The day after implantation, the mice started to receive CD44-3MUT-Fc, control (rhIgG1-Fc) or vehicle (PBS) every second day via IP injections for 14 days. ( A ) Schematic presentation of experimental design. ( B ) Relative blood vessel invasion into matrix–filled angioreactors. The data are represented as the mean ± SEM. Datapoints show the mean of 4 angioreactors for individual mice. N – the number of independent experiments. GF – growth factors (FGF-2/VEGF). ( C ) Effect size with 95% confidence intervals (upper row) and P values (lower row) of pairwise comparisons of the data shown in ( B ). Effect sizes were calculated by Cohen’s d formula. Confidence intervals were derived using bootstrap resampling. P values are from t tests using pooled SD.

Article Snippet: After blocking, samples and standards (Recombinant Mouse CD44 Fc Chimera; R&D Systems) were incubated in wells for 30 min at 37°C.

Techniques: Recombinant, In Vivo, Quantitation Assay, Control, Derivative Assay

Journal: bioRxiv

Article Title: CD44 Controls Endothelial Proliferation and Functions as Endogenous Inhibitor of Angiogenesis

doi: 10.1101/049494

Figure Lengend Snippet: Soluble CD44 concentrations in mouse serum. Blood was collected from mice of different genetic backgrounds and the mice used in the angiogenesis experiments shown in and . ( A ) Serum levels of soluble CD44 in mice from different strains. Each dot represents an individual mouse. Cross indicates the mean. P values are from ANOVA post hoc comparisons using the Tukey HSD test. ( B ) The correlation between relative blood vessel invasion and post-experiment serum sCD44 in wild-type mice. Cd44 -null mice and nude mice were excluded from the dataset. Pearson’s r and the associated P values are shown. ( C ) Post-experiment serum levels of sCD44 in nude mice from different treatment groups. Treatments where more than five mice were analysed are shown. Each dot represents an individual mouse. Cross indicates the mean. GF – growth factors (FGF-2/VEGF). ( D ) The correlation between relative blood vessel invasion and post-experiment serum sCD44 in nude mice. Pearson’s r and the associated P values are shown. In ( B and D ), dashed line is the linear model fit, gray shading is the standard error interval of fitted values.

Article Snippet: After blocking, samples and standards (Recombinant Mouse CD44 Fc Chimera; R&D Systems) were incubated in wells for 30 min at 37°C.

Techniques:

Journal: bioRxiv

Article Title: CD44 Controls Endothelial Proliferation and Functions as Endogenous Inhibitor of Angiogenesis

doi: 10.1101/049494

Figure Lengend Snippet: CD44-3MUT-Fc inhibits EC growth. ( A ) Real-time track of cell adhesion and synchronisation of HUVECs seeded onto 96-well electrode arrays. After seeding, the cells were grown for about 24 h. After that the cells were starved overnight in the media supplemented with 1% FBS (gray area). 1 h before the release from serum starvation (vertical dashed line) the cells were preincubated with different concentrations of rhIgG-Fc or CD44-3MUT-Fc in 5% FBS–containing media. ( B ) Growth curves of HUVECs released from serum starvation by supplementing preincubation media with 25 ng/ml VEGF. Facet labels show rhIgG-Fc or CD44-3MUT-Fc concentrations during preincubation. The data are represented as the mean ± SEM. N – the number of independent experiments. ( C–F ) HUVECs were synchronised and pretreated as in panel A. After preincubation, the cells were stimulated either with 25 ng/ml FGF-2 ( C ), 25 ng/ml VEGF ( D ), 63 ng/ml HGF ( E ) or 10 ng/ml GDF-2 ( F ). After 72 h, the number of viable cells was quantitated by measuring the ATP per well. Left: the effect of growth factor stimulation and the effect of 10 nM fumagillin (FUM) as a positive control for inhibition of cell proliferation. Right: the dose-response curves of rhIgG-Fc (filled triangles) and CD44-3MUT-Fc (filled circles). The data are represented as the mean ± SEM. N = 3–4 independent experiments. ( G ) CD44-3MUT-Fc dose-response curves for FGF-2, GDF-2, HGF or VEGF stimulated HUVEC. The data are represented as the mean ± SEM. ( H ) Apoptosis of HUVECs stimulated with different growth factors and treated with 12.64 μM (-4.9 log 10 M) rhIgG-Fc, CD44-3MUT-Fc or left untreated. Apoptosis was quantitated by Annexin V staining. The data are represented as the mean ± SEM. N = 2 independent experiments. P values are from the ANOVA post hoc comparisons using the Tukey HSD test.

Article Snippet: After blocking, samples and standards (Recombinant Mouse CD44 Fc Chimera; R&D Systems) were incubated in wells for 30 min at 37°C.

Techniques: Positive Control, Inhibition, Staining

Journal: bioRxiv

Article Title: CD44 Controls Endothelial Proliferation and Functions as Endogenous Inhibitor of Angiogenesis

doi: 10.1101/049494

Figure Lengend Snippet: CD44 knockdown augments EC growth. siRNA transfected HUVECs were plated onto 96-well electrode arrays. After 24 h, the cells were starved in 1% FBS media overnight. After starving, the cells were released from cell cycle block by the addition of 20% FBS (A) ; 8 ng/ml, 25 ng/ml, or 79 ng/ml FGF-2 or VEGF (C) ; and 2.5 ng/ml or 10 ng/ml GDF-2 (E) . Following stimulation, HUVEC growth was monitored by recording electrode impedance. Raw impedance readings are shown to allow direct comparison to endpoint measurements. N = 2 for 5% and 20% FBS, 25 ng/ml and 79 ng/ml FGF-2, and 8 ng/ml and 25 ng/ml VEGF. (B, D, F) 72 h after release from cell cycle block the viable cell numbers were determined by measuring the ATP per well. Treatments are labeled as shown in ( B ). The data are represented as the mean ± SEM. P values are from the ANOVA post hoc comparisons using the Tukey HSD test. P values ≤ 0.05 of siCD44-siNTP comparisons are shown. siCD44-siNTP comparisons: N = 4 for 2.5 ng/ml and 10 ng/ml GDF-2, and N = 5 for 20% FBS, 25 ng/ml and 79 ng/ml FGF-2, and 8 ng/ml and 25 ng/ml VEGF. (G) Western blot analysis of CD44 silencing in HUVECs transfected with 30 nM siRNAs for 48 h. siNTP – non-targeting siRNA pool, siVIM – vimentin-targeting pool, siCD44 – CD44-targeting pool, UT – non-transfected cells.

Article Snippet: After blocking, samples and standards (Recombinant Mouse CD44 Fc Chimera; R&D Systems) were incubated in wells for 30 min at 37°C.

Techniques: Knockdown, Transfection, Blocking Assay, Comparison, Labeling, Western Blot

Journal: Nutrients

Article Title: Oligosaccharides Ameliorate Acute Kidney Injury by Alleviating Cluster of Differentiation 44-Mediated Immune Responses in Renal Tubular Cells

doi: 10.3390/nu14040760

Figure Lengend Snippet: Inhibitory effect of oligosaccharides on CD44 signal transduction in hypoxic NRK-52E cells. The cells were pretreated with FC, FOS, or GOS for 30 min and then subjected to hypoxic culture. Protein expression was analyzed by Western blotting. ( A ) Dose-dependent effects of oligosaccharides on CD44 expression. ( B ) Effect of oligosaccharides on CD44-related signals. Oligosaccharide concentration was set as 0.1 mg/mL. Relative increases in protein bands are also presented in bar chart form ( C ). Results are expressed as mean ± SD (n = 4).

Article Snippet: Recombinant rat CD44 was obtained from R&D Systems (Minneapolis, MN, USA, Cat. No. 6577-CD) and employed to produce a binding reaction with HA in accordance with the manufacturer’s instructions.

Techniques: Transduction, Expressing, Western Blot, Concentration Assay

Journal: Nutrients

Article Title: Oligosaccharides Ameliorate Acute Kidney Injury by Alleviating Cluster of Differentiation 44-Mediated Immune Responses in Renal Tubular Cells

doi: 10.3390/nu14040760

Figure Lengend Snippet: Role of CD44 and JNK in hypoxia-induced inflammatory responses in NRK-52E cells. Protein expression was analyzed by Western blot. ( A ) Inhibitory effect of CD44 siRNA transfection on phosphorylated JNK and cytokines. ( B ) Inhibitory effect of SP600125 on cytokines. The cells were pretreated with 20 μM SP600125 for 30 min prior to hypoxia treatment. Relative increases in protein bands are also presented in bar chart form. Results are expressed as mean ± SD (n = 4).

Article Snippet: Recombinant rat CD44 was obtained from R&D Systems (Minneapolis, MN, USA, Cat. No. 6577-CD) and employed to produce a binding reaction with HA in accordance with the manufacturer’s instructions.

Techniques: Expressing, Western Blot, Transfection

Journal: Nutrients

Article Title: Oligosaccharides Ameliorate Acute Kidney Injury by Alleviating Cluster of Differentiation 44-Mediated Immune Responses in Renal Tubular Cells

doi: 10.3390/nu14040760

Figure Lengend Snippet: Competitiveness of oligosaccharides and HA in binding to CD44. ( A ) Reducing effect of oligosaccharides on CD44 antigenicity. Recombinant rat CD44 (20 ng/mL) was mixed with or without FC, FOS, or GOS for 10 min and then analyzed using the CD44 ELISA kit. Results are expressed as mean ± SD (n = 4). *, p < 0.05 vs. the control group. ( B ) HA interfering with the inhibitory effect of oligosaccharides on CD44 and phosphorylated JNK expression. NRK-52E cells were pretreated with 0.1 mg/mL HA for 30 min, then administered with 0.1 mg/mL FC, FOS, or GOS for 30 min and finally subjected to hypoxic culture. Protein expression was analyzed by Western blot. Relative increases in the protein bands are also presented in bar chart form. Results are expressed as mean ± SD (n = 4).

Article Snippet: Recombinant rat CD44 was obtained from R&D Systems (Minneapolis, MN, USA, Cat. No. 6577-CD) and employed to produce a binding reaction with HA in accordance with the manufacturer’s instructions.

Techniques: Binding Assay, Recombinant, Enzyme-linked Immunosorbent Assay, Control, Expressing, Western Blot

Journal: Bioactive materials

Article Title: Advanced bioactive glue tethering Lubricin/PRG4 to promote integrated healing of avascular meniscus tears.

doi: 10.1016/j.bioactmat.2023.04.026

Figure Lengend Snippet: Fig. 7. CD44, a transmembrane glycoprotein, has a reported binding affinity to HA, lubricin, and fibrin(ogen) (A). CD44 readily bound on the articular surface of meniscus where lubricin was present, but not on NaCl-treated surface with depleted lubricin (B).

Article Snippet: Similarly, CD44 Fc chimera protein (10 μg/mL, R&D Systems, Minneapolis, MN) was coated, followed by crosslinking between protein G and CD44 using bis(sulfosuccinimidyl) suberate (50 mM BS3, Thermo Fisher Scientific, Waltham, MA).

Techniques: Binding Assay

Journal: Bioactive materials

Article Title: Advanced bioactive glue tethering Lubricin/PRG4 to promote integrated healing of avascular meniscus tears.

doi: 10.1016/j.bioactmat.2023.04.026

Figure Lengend Snippet: Fig. 8. Heparin conjugated FibGen incorporated with CD44 (Hep-FibGen-CD44) (A) showed significantly higher lap shear modulus and strength compared to FibGen on lubricin-coated meniscal tissues, with no significant difference from FibGen-CD44 (B) (*:p < 0.001; n = 10 per group). In contrast, Hep-FibGen-CD44 showed significantly higher lap shear and modulus as compared to Hep-FibGen on HA/lubricin-coated meniscal tissues (C) (*:p < 0.001 compared to FibGen, #:p < 0.001 compared to Hep-FibGen; n = 10 per group). Histologically, delivery of CTGF and TGFβ3-μS via Hep-FibGen-CD44 and Hep-FibGen improved the healing of avascular meniscus tears as compared to FibGen (D) (dash-line: healing zone). Quantitative histomorphometry measurements show significant improvement of tissue integration with FibGen-CD44 and Hep-FibGen-CD44 (E) (*:p < 0.001 compared to FibGen, #:p < 0.001 compared to FibGen-CD44; n = 5 per group). By 6 weeks, the tensile modulus and strength (F) of healed meniscus were significantly higher with CD44 incorporation and heparin conjugation than FibGen (*:p < 0.001 compared to FibGen, #:p < 0.001 compared to FibGen-CD44; n = 6–7 per group).

Article Snippet: Similarly, CD44 Fc chimera protein (10 μg/mL, R&D Systems, Minneapolis, MN) was coated, followed by crosslinking between protein G and CD44 using bis(sulfosuccinimidyl) suberate (50 mM BS3, Thermo Fisher Scientific, Waltham, MA).

Techniques: Shear, Conjugation Assay

Journal: Bioactive materials

Article Title: Advanced bioactive glue tethering Lubricin/PRG4 to promote integrated healing of avascular meniscus tears.

doi: 10.1016/j.bioactmat.2023.04.026

Figure Lengend Snippet: Fig. 9. Speculated mechanism for synergistic effect of heparin conjugation and CD44 incorporation on yielding strong binding to HA- and lubricin-coated meniscus surface. Although specific binding domain, mechanism, and affinity between each other components have been incompletely described, the cross- binding affinity between HA, lubricin, fibrinogen, heparin, and CD44 may have potential to achieve a strong initial binding of our bioactive glue on HA/ lubricin-deposited meniscus surface.

Article Snippet: Similarly, CD44 Fc chimera protein (10 μg/mL, R&D Systems, Minneapolis, MN) was coated, followed by crosslinking between protein G and CD44 using bis(sulfosuccinimidyl) suberate (50 mM BS3, Thermo Fisher Scientific, Waltham, MA).

Techniques: Conjugation Assay, Binding Assay

Journal: bioRxiv

Article Title: CD44 Controls Endothelial Proliferation and Functions as Endogenous Inhibitor of Angiogenesis

doi: 10.1101/049494

Figure Lengend Snippet: Increased angiogenesis in CD44-null mice. ( A ) Angiogenesis was analysed in C3H, C57BL/6, and in wild-type and Cd44 −/− mice of mixed genetic backgrounds (WT mix and Cd44KO mix, respectively). Basement membrane extract–filled angioreactors containing premixed FGF-2, VEGF and heparin or PBS for uninduced controls were implanted SC into the flanks of mice. Each mouse received 2 angioreactors, 1 per flank. 14 days after implantation, angioreactors were resected and the population of ECs within the angioreactor matrix was assessed by FITC-lectin staining. The number of fluorescent cells was quantitated by microplate reader. Raw readings from independent experiments were scaled by dividing by their quadratic mean. N = 2–5 mice per condition from 2 independent experiments. P value is from Student’s t-test. ( B ) Angiogenesis in Cd44 −/− mice and their heterozygous ( Cd44 +/− ) and wild-type ( Cd44 +/+ ) littermate that had been backcrossed six generations to the C57BL/6 background. The data are represented as the mean ± SEM. Each dot represents the mean of two angioreactors for an individual mouse. N = 8 mice per condition from 2 independent experiments. P values are from ANOVA post hoc comparisons using the Tukey HSD test.

Article Snippet: CD44-3MUT with C-terminal human IgG1-Fc domain, recombinant human IgG1-Fc domain (rhIgG-Fc) and irrelevant human IgG1 mAb were produced by Icosagen Cell Factory (Estonia).

Techniques: Staining

Journal: bioRxiv

Article Title: CD44 Controls Endothelial Proliferation and Functions as Endogenous Inhibitor of Angiogenesis

doi: 10.1101/049494

Figure Lengend Snippet: Recombinant CD44-3MUT Fc fusion protein inhibits angiogenesis in vivo . First, the assay and quantitation were performed similarly to those described in , except that each mouse received 4 angioreactors, 2 per flank. The day after implantation, the mice started to receive CD44-3MUT-Fc, control (rhIgG1-Fc) or vehicle (PBS) every second day via IP injections for 14 days. ( A ) Schematic presentation of experimental design. ( B ) Relative blood vessel invasion into matrix–filled angioreactors. The data are represented as the mean ± SEM. Datapoints show the mean of 4 angioreactors for individual mice. N – the number of independent experiments. GF – growth factors (FGF-2/VEGF). ( C ) Effect size with 95% confidence intervals (upper row) and P values (lower row) of pairwise comparisons of the data shown in ( B ). Effect sizes were calculated by Cohen’s d formula. Confidence intervals were derived using bootstrap resampling. P values are from t tests using pooled SD.

Article Snippet: CD44-3MUT with C-terminal human IgG1-Fc domain, recombinant human IgG1-Fc domain (rhIgG-Fc) and irrelevant human IgG1 mAb were produced by Icosagen Cell Factory (Estonia).

Techniques: Recombinant, In Vivo, Quantitation Assay, Derivative Assay

Journal: bioRxiv

Article Title: CD44 Controls Endothelial Proliferation and Functions as Endogenous Inhibitor of Angiogenesis

doi: 10.1101/049494

Figure Lengend Snippet: Soluble CD44 concentrations in mouse serum. Blood was collected from mice of different genetic backgrounds and the mice used in the angiogenesis experiments shown in and . ( A ) Serum levels of soluble CD44 in mice from different strains. Each dot represents an individual mouse. Cross indicates the mean. P values are from ANOVA post hoc comparisons using the Tukey HSD test. ( B ) The correlation between relative blood vessel invasion and post-experiment serum sCD44 in wild-type mice. Cd44 -null mice and nude mice were excluded from the dataset. Pearson’s r and the associated P values are shown. ( C ) Post-experiment serum levels of sCD44 in nude mice from different treatment groups. Treatments where more than five mice were analysed are shown. Each dot represents an individual mouse. Cross indicates the mean. GF – growth factors (FGF-2/VEGF). ( D ) The correlation between relative blood vessel invasion and post-experiment serum sCD44 in nude mice. Pearson’s r and the associated P values are shown. In ( B and D ), dashed line is the linear model fit, gray shading is the standard error interval of fitted values.

Article Snippet: CD44-3MUT with C-terminal human IgG1-Fc domain, recombinant human IgG1-Fc domain (rhIgG-Fc) and irrelevant human IgG1 mAb were produced by Icosagen Cell Factory (Estonia).

Techniques:

Journal: bioRxiv

Article Title: CD44 Controls Endothelial Proliferation and Functions as Endogenous Inhibitor of Angiogenesis

doi: 10.1101/049494

Figure Lengend Snippet: CD44-3MUT-Fc inhibits EC growth. ( A ) Real-time track of cell adhesion and synchronisation of HUVECs seeded onto 96-well electrode arrays. After seeding, the cells were grown for about 24 h. After that the cells were starved overnight in the media supplemented with 1% FBS (gray area). 1 h before the release from serum starvation (vertical dashed line) the cells were preincubated with different concentrations of rhIgG-Fc or CD44-3MUT-Fc in 5% FBS–containing media. ( B ) Growth curves of HUVECs released from serum starvation by supplementing preincubation media with 25 ng/ml VEGF. Facet labels show rhIgG-Fc or CD44-3MUT-Fc concentrations during preincubation. The data are represented as the mean ± SEM. N – the number of independent experiments. ( C–F ) HUVECs were synchronised and pretreated as in panel A. After preincubation, the cells were stimulated either with 25 ng/ml FGF-2 ( C ), 25 ng/ml VEGF ( D ), 63 ng/ml HGF ( E ) or 10 ng/ml GDF-2 ( F ). After 72 h, the number of viable cells was quantitated by measuring the ATP per well. Left: the effect of growth factor stimulation and the effect of 10 nM fumagillin (FUM) as a positive control for inhibition of cell proliferation. Right: the dose-response curves of rhIgG-Fc (filled triangles) and CD44-3MUT-Fc (filled circles). The data are represented as the mean ± SEM. N = 3–4 independent experiments. ( G ) CD44-3MUT-Fc dose-response curves for FGF-2, GDF-2, HGF or VEGF stimulated HUVEC. The data are represented as the mean ± SEM. ( H ) Apoptosis of HUVECs stimulated with different growth factors and treated with 12.64 μM (-4.9 log 10 M) rhIgG-Fc, CD44-3MUT-Fc or left untreated. Apoptosis was quantitated by Annexin V staining. The data are represented as the mean ± SEM. N = 2 independent experiments. P values are from the ANOVA post hoc comparisons using the Tukey HSD test.

Article Snippet: CD44-3MUT with C-terminal human IgG1-Fc domain, recombinant human IgG1-Fc domain (rhIgG-Fc) and irrelevant human IgG1 mAb were produced by Icosagen Cell Factory (Estonia).

Techniques: Positive Control, Inhibition, Staining

Journal: bioRxiv

Article Title: CD44 Controls Endothelial Proliferation and Functions as Endogenous Inhibitor of Angiogenesis

doi: 10.1101/049494

Figure Lengend Snippet: CD44 knockdown augments EC growth. siRNA transfected HUVECs were plated onto 96-well electrode arrays. After 24 h, the cells were starved in 1% FBS media overnight. After starving, the cells were released from cell cycle block by the addition of 20% FBS (A) ; 8 ng/ml, 25 ng/ml, or 79 ng/ml FGF-2 or VEGF (C) ; and 2.5 ng/ml or 10 ng/ml GDF-2 (E) . Following stimulation, HUVEC growth was monitored by recording electrode impedance. Raw impedance readings are shown to allow direct comparison to endpoint measurements. N = 2 for 5% and 20% FBS, 25 ng/ml and 79 ng/ml FGF-2, and 8 ng/ml and 25 ng/ml VEGF. (B, D, F) 72 h after release from cell cycle block the viable cell numbers were determined by measuring the ATP per well. Treatments are labeled as shown in ( B ). The data are represented as the mean ± SEM. P values are from the ANOVA post hoc comparisons using the Tukey HSD test. P values ≤ 0.05 of siCD44-siNTP comparisons are shown. siCD44-siNTP comparisons: N = 4 for 2.5 ng/ml and 10 ng/ml GDF-2, and N = 5 for 20% FBS, 25 ng/ml and 79 ng/ml FGF-2, and 8 ng/ml and 25 ng/ml VEGF. (G) Western blot analysis of CD44 silencing in HUVECs transfected with 30 nM siRNAs for 48 h. siNTP – non-targeting siRNA pool, siVIM – vimentin-targeting pool, siCD44 – CD44-targeting pool, UT – non-transfected cells.

Article Snippet: CD44-3MUT with C-terminal human IgG1-Fc domain, recombinant human IgG1-Fc domain (rhIgG-Fc) and irrelevant human IgG1 mAb were produced by Icosagen Cell Factory (Estonia).

Techniques: Transfection, Blocking Assay, Labeling, Western Blot