Journal: JCI Insight

Article Title: Dysregulated fibrinolysis and plasmin activation promote the pathogenesis of osteoarthritis

doi: 10.1172/jci.insight.173603

Figure Lengend Snippet: ( A ) Representative images from immunofluorescence staining of plasmin (green, left), staining of nuclei (blue, middle), and merging (right) in the undamaged articular cartilage area from individuals with knee OA who underwent total knee replacement. ( B ) Representative images from immunohistochemical staining of plasmin in the damaged articular cartilage area (upper left), the synovium (upper right), and the isotype controls (bottom, respectively) from individuals with OA. The arrowhead indicates binding of plasmin on the surface of chondrocytes (upper left) and cells of the synovial lining (upper right). ( A and B ) Scale bar, 200 μm; cartilage and synovial tissues from n = 5 individuals were analyzed. ( C ) Degradation of recombinant lubricin, shown on SDS-PAGE gel stained with Coomassie blue, by plasmin, but not activated tPA or uPA after 4 hours’ 37°C incubation. Red arrowhead shows the lubricin stained with Coomassie blue in different conditions: vehicle, plasmin, tPA, and uPA. ( D ) ELISA quantification of soluble sGAG released from cartilage explants from individuals with OA, treated with vehicle, pro–MMP-13, plasmin, or plasmin + pro–MMP-13. ( E ) Quantitative PCR (qPCR) analysis of OA-related inflammatory and degradative mediators as well as VEGFα in human primary synoviocytes, derived from the knee joints of individuals with OA, with or without plasmin stimulation. ( F and G ) qPCR analysis of relative gene expression levels of OA-related inflammatory and degradative mediators in synovial tissue ( F ) or articular cartilage from Plg +/+ ( n = 5) and Plg –/– ( n = 5) mice 20 weeks after DMM. All data are the mean ± SEM of triplicates and are representative of 3 independent experiments. ** P < 0.01, and *** P < 0.001. The test in panel D is 1-way ANOVA. The test in panels E – G is 2-tailed t test.

Article Snippet: Human recombinant lubricin (20 μg) (gift from Lubris Biopharma, Weston, Massachusetts, USA) was incubated at 37°C with (i) recombinant human plasmin, (ii) tPA, and (iii) uPA for 4 hours.

Techniques: Immunofluorescence, Staining, Immunohistochemical staining, Binding Assay, Recombinant, SDS Page, Incubation, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction, Derivative Assay, Gene Expression

Journal: Frontiers in Molecular Biosciences

Article Title: Truncated lubricin glycans in osteoarthritis stimulate the synoviocyte secretion of VEGFA, IL-8, and MIP-1 α : Interplay between O -linked glycosylation and inflammatory cytokines

doi: 10.3389/fmolb.2022.942406

Figure Lengend Snippet: Determination of Tn and T antigens on SF-lubricin. (A) Example of LC-SRM MS of released Tn antigen measured as GalNAcol and T antigen measured as Galβ1-3-GalNAcol from an OA patient. (B) Example of LC-SRM MS of GalNAcol and Galβ1-3-GalNAcol from standards. (C) Correlation between Tn and T antigens using MS or lectins, where the level of Tn antigen (GalNAcα1-Ser/Thr) was measured using the lectin HAA and the level of T antigen (Galβ1-3GalNAcα1-Ser/Thr was measured using the PNA lectin using patients diagnosed with OA (n = 16). Spearman correlation r and significance P were calculated and displayed in the diagram. (D) Absorbances of HAA-epitopes/PNA-epitopes on SF lubricin were calculated from 29 OA patients and 16 controls using lectin ELISA. Significance was calculated by a two-tailed nonparametric Mann–Whitney test. Outliers were excluded from prior calculations according to the GOUT test with Q = 0.1. Cartoons of monosaccharide building blocks for representing oligosaccharides according to the SNFG nomenclature; yellow circle = Gal and yellow square = GalNAc.

Article Snippet: Recombinant human lubricin (rhPRG4, Lubris BioPharma, Framingham, MA) was isolated as described ( ; ) and used as a standard.

Techniques: Enzyme-linked Immunosorbent Assay, Two Tailed Test, MANN-WHITNEY

Journal: Frontiers in Molecular Biosciences

Article Title: Truncated lubricin glycans in osteoarthritis stimulate the synoviocyte secretion of VEGFA, IL-8, and MIP-1 α : Interplay between O -linked glycosylation and inflammatory cytokines

doi: 10.3389/fmolb.2022.942406

Figure Lengend Snippet: SF-lubricin glycan modification in OA increases the inflammatory biomarker expression. Left-hand panels (A–F) show correlations between HAA/PNA ratio and inflammatory cytokines in SF samples from OA patients (red dots, n = 29), also including non-OA SF-controls (blue dots, n = 2). Outliers were excluded from prior calculations according to the GOUT test with Q = 0.1. Spearman correlation (r S ) and p -values are displayed in each diagram. Right-hand panels (G–L) show cytokine concentrations in the medium (mean ± SD) secreted from fibroblast-like synoviocytes (FLSs) from healthy subjects ( n = 3, blue dots) and from OA patients ( n = 3, red dots) after treatment of recombinant lubricin (rhPRG4) with or without treatment with β-galactosidase and sialidase. In L, the main oligosaccharides present on rhPRG4 shown with or without digestion corresponding to (from left) “only enzymes” means FLSs treated with PBS, including sialidase and β -galactosidase without rhPRG4, “T + sialyl T″ FLSs treated with intact rhPRG4, “T” FLSs treated with rhPRG4 subjected to sialidase, “Tn + sialyl T″ FLSs treated with rhPRG4 subjected to β -galactosidase, and “Tn” FLSs treated with rhPRG4 subjected to both sialidase and β-galactosidase. Cartoons of monosaccharide building blocks for representing oligosaccharides on lubricin according to the SNFG nomenclature; yellow circle = Gal, yellow square = GalNAc, and purple diamond = NeuAc. For statistics, multiple comparison and one-way ANOVA were used. ns = not significant, * indicates p < 0.05, ** p < 0.01, *** p < 0.001, and ****<0.0001. Secretion of cytokines from controls’ and OA patients’ FLSs subjected to only PBS showed no statistically significant differences compared to treating the FLSs with intact rhPRG4 or “only enzymes” (data not shown).

Article Snippet: Recombinant human lubricin (rhPRG4, Lubris BioPharma, Framingham, MA) was isolated as described ( ; ) and used as a standard.

Techniques: Glycoproteomics, Modification, Biomarker Discovery, Expressing, Recombinant, Comparison

Journal: Frontiers in Molecular Biosciences

Article Title: Truncated lubricin glycans in osteoarthritis stimulate the synoviocyte secretion of VEGFA, IL-8, and MIP-1 α : Interplay between O -linked glycosylation and inflammatory cytokines

doi: 10.3389/fmolb.2022.942406

Figure Lengend Snippet: Lubricin glycosylation in OA. (A) Schematic representation of mucin-type lubricin with its glycans in OA and in normal conditions based on data from this report and previous ones (not displaying all of the 172 glycosylation sites of lubricin ). (B) Glyco-mediated OA inflammation where pathological OA Tn glycoforms of lubricin are interacting with unknown Tn receptors to trigger specific cytokine secretion. The figure was made using BioRender ( www.biorender.com ).

Article Snippet: Recombinant human lubricin (rhPRG4, Lubris BioPharma, Framingham, MA) was isolated as described ( ; ) and used as a standard.

Techniques: Glycoproteomics

Journal: ACS Applied Materials & Interfaces

Article Title: A Mucin-Deficient Ocular Surface Mimetic Platform for Interrogating Drug Effects on Biolubrication, Antiadhesion Properties, and Barrier Functionality

doi: 10.1021/acsami.1c22280

Figure Lengend Snippet: Mucin-specific protease StcE-induced frictional damage at model cornea–palpebral conjunctiva and bulbar conjunctiva–palpebral conjunctiva interfaces. (A) Idealized schematic of the LCR setup for the model cornea–palpebral conjunctiva interface immersed in cell culture medium, in which differentiated HCjE cells on the top plate laterally shear differentiated hTCEpi cells on the bottom plate. (B) Representative image showing the shearing of hTCEpi cells following a 5 μm displacement of the top plate, with green and orange dashed lines highlighting cell boundaries before displacement (not otherwise shown) and after displacement, respectively. (C) Representative LCR stress relaxation curve for the model cornea–palpebral conjunctiva interface with and without StcE treatment. (D, E) Peak and residual moduli for LCR experiments shearing (D) control and StcE-treated differentiated HCjE cells against control and StcE-treated hTCEpi cells (data: mean ± SD, n = 11 (control), 13 (StcE)) or (E) control and StcE-treated differentiated HCjE cells against control and StcE-treated differentiated HCjE cells (data: mean ± SE, n = 12). Statistical significance ( p < 0.05) was determined using two-tailed Welch’s t -tests. (F, G) Lubricin supplementation attenuated the increase in peak and residual moduli observed when the differentiated cell layers were StcE treated at the (F) model cornea–palpebral conjunctiva but not at the (G) model bulbar conjunctiva–palpebral conjunctiva interface (data = mean ± SD, n = 9–13). Statistical significance ( p < 0.05) was determined using one-way Welch's ANOVA with post-hoc Dunnett’s T3 multiple comparisons testing relative to StcE-treated (0 μg/mL) cells.

Article Snippet: Recombinant human lubricin was generously provided by Novartis (Basel, Switzerland) at a concentration of 2.01 mg/mL (including O -glycosylation) in 10 mM sodium phosphate, 140 mM sodium chloride, and 0.02% (w/v) polysorbate 20, pH 7.0.

Techniques: Cell Culture, Shear, Control, Two Tailed Test

Journal: ACS Applied Materials & Interfaces

Article Title: A Mucin-Deficient Ocular Surface Mimetic Platform for Interrogating Drug Effects on Biolubrication, Antiadhesion Properties, and Barrier Functionality

doi: 10.1021/acsami.1c22280

Figure Lengend Snippet: Induced mucin deficiency increased rose bengal penetrance for differentiated hTCEpi and HCjE cells, an effect attenuated by lubricin addition. (A) Rose bengal staining in differentiated HCjE cells. Islands of rose bengal-negative cells appeared after 7 days in stratification medium. StcE-induced mucin deficiency increased dye penetrance, which was reversed following 2 h supplementation with 25 μg/mL recombinant human lubricin. (B, C) Effect of overnight StcE treatment on rose bengal uptake for (B) hTCEpi and (C) HCjE cells. (D, E) Effect of 25 μg/mL lubricin supplementation on rose bengal uptake in StcE-treated (0 μg/mL) hTCEpi (D) and HCjE (E) cells. Statistical significance ( p < 0.05) was determined using a two-tailed Welch’s t -test.

Article Snippet: Recombinant human lubricin was generously provided by Novartis (Basel, Switzerland) at a concentration of 2.01 mg/mL (including O -glycosylation) in 10 mM sodium phosphate, 140 mM sodium chloride, and 0.02% (w/v) polysorbate 20, pH 7.0.

Techniques: Staining, Recombinant, Two Tailed Test

Journal: ACS Applied Materials & Interfaces

Article Title: A Mucin-Deficient Ocular Surface Mimetic Platform for Interrogating Drug Effects on Biolubrication, Antiadhesion Properties, and Barrier Functionality

doi: 10.1021/acsami.1c22280

Figure Lengend Snippet: Induced mucin deficiency reduced the antiadhesive character of differentiated hTCEpi and HCjE cells, an effect attenuated by lubricin addition. (A, B) CellTracker Deep Red-labeled HCjE cells in suspension showed significantly increased binding to differentiated (A) hTCEpi and (B) HCjE cells following StcE treatment in both cell culture medium (NT) and cell culture medium containing vehicle (Vehicle) (data = mean ± SD, n = 4). Statistical significance ( p < 0.05) was determined using two-tailed Welch’s t -tests. (C, D) Binding of CellTracker Deep Red-labeled HCjE cells in suspension was not significantly affected by the presence of vehicle for control cells or StcE-treated cells (data = mean ± SD, n = 4). Statistical significance ( p < 0.05) was determined using two-tailed Welch’s t -tests. (E, F) Supplementation with recombinant human lubricin restored the antiadhesive character of StcE-treated (E) hTCEpi and (F) HCjE cells (data = mean ± SD, n = 4). Statistical significance ( p < 0.05) was determined using one-way Welch's ANOVA with post-hoc Dunnett’s T3 multiple comparisons testing relative to StcE-treated (0 μg/mL) cells.

Article Snippet: Recombinant human lubricin was generously provided by Novartis (Basel, Switzerland) at a concentration of 2.01 mg/mL (including O -glycosylation) in 10 mM sodium phosphate, 140 mM sodium chloride, and 0.02% (w/v) polysorbate 20, pH 7.0.

Techniques: Labeling, Suspension, Binding Assay, Cell Culture, Two Tailed Test, Control, Recombinant

Journal: ACS Applied Materials & Interfaces

Article Title: A Mucin-Deficient Ocular Surface Mimetic Platform for Interrogating Drug Effects on Biolubrication, Antiadhesion Properties, and Barrier Functionality

doi: 10.1021/acsami.1c22280

Figure Lengend Snippet: Recombinant human lubricin exhibited dose-dependent biolubrication properties at model mucin-deficient cornea–palpebral conjunctiva interfaces. (A) Peak modulus exhibited a concentration-dependent response ( p < 0.0029) to lubricin supplementation (data = mean ± SE, n = 7–13). (B) Lubricin supplementation also reduced the residual modulus in a dose-dependent manner ( p < 0.0011) (data = mean ± SE, n = 7–13). (C) Idealized schematic showing possible mechanism behind observed dose-dependent biolubrication effects at dry eye mimetic ocular surfaces. Statistical significance ( p < 0.05) was determined using one-way Welch's ANOVA (A, B).

Article Snippet: Recombinant human lubricin was generously provided by Novartis (Basel, Switzerland) at a concentration of 2.01 mg/mL (including O -glycosylation) in 10 mM sodium phosphate, 140 mM sodium chloride, and 0.02% (w/v) polysorbate 20, pH 7.0.

Techniques: Recombinant, Concentration Assay

Journal: ACS Applied Materials & Interfaces

Article Title: A Mucin-Deficient Ocular Surface Mimetic Platform for Interrogating Drug Effects on Biolubrication, Antiadhesion Properties, and Barrier Functionality

doi: 10.1021/acsami.1c22280

Figure Lengend Snippet: Lubricin molecules subjected to accelerated aging conditions still exhibited dose-dependent biolubrication properties at model cornea–palpebral conjunctiva interfaces. Peak (A–D) and residual (E–H) modulus values for StcE-treated model cornea–palpebral conjunctiva interfaces in the presence of (A, E) unstressed drug substance and (B, F) temperature-stressed, (C, G) acid-stressed, and (D, H) alkaline-stressed lubricin (I, J) at 0, 25, 75, and 250 μg/mL. Stressed lubricin molecules exhibited attenuated biolubrication properties at 25 μg/mL, with significantly higher (I) peak and (J) residual modulus values observed. Statistical significance ( p < 0.05) was determined using one-way Welch's ANOVA with post-hoc Dunnett’s T3 multiple comparisons testing relative to StcE-treated (0 μg/mL) cells (A–H) or unstressed drug substance (DS) (I, J).

Article Snippet: Recombinant human lubricin was generously provided by Novartis (Basel, Switzerland) at a concentration of 2.01 mg/mL (including O -glycosylation) in 10 mM sodium phosphate, 140 mM sodium chloride, and 0.02% (w/v) polysorbate 20, pH 7.0.

Techniques:

Journal: ACS Applied Materials & Interfaces

Article Title: A Mucin-Deficient Ocular Surface Mimetic Platform for Interrogating Drug Effects on Biolubrication, Antiadhesion Properties, and Barrier Functionality

doi: 10.1021/acsami.1c22280

Figure Lengend Snippet: Lubricin subjected to accelerated aging conditions still exhibited antiadhesive properties on StcE-treated differentiated hTCEpi cells. Supplementation with unstressed recombinant human lubricin (A) and lubricin subjected to temperature stress (B), acid stress (C), or alkaline stress (D) reduced the adhesion of CellTracker Deep Red-labeled HCjE cells in suspension to StcE-treated hTCEpi cells (data = mean ± SD, n = 4). (E) Subjecting lubricin to accelerated aging conditions resulted in more variable antiadhesive properties. Statistical significance ( p < 0.05) was determined using one-way Welch's ANOVA with post-hoc Dunnett’s T3 multiple comparisons testing relative to StcE-treated (0 μg/mL) cells (A–D) or unstressed drug substance (DS) (E).

Article Snippet: Recombinant human lubricin was generously provided by Novartis (Basel, Switzerland) at a concentration of 2.01 mg/mL (including O -glycosylation) in 10 mM sodium phosphate, 140 mM sodium chloride, and 0.02% (w/v) polysorbate 20, pH 7.0.

Techniques: Recombinant, Labeling, Suspension

Journal: ACS Applied Materials & Interfaces

Article Title: A Mucin-Deficient Ocular Surface Mimetic Platform for Interrogating Drug Effects on Biolubrication, Antiadhesion Properties, and Barrier Functionality

doi: 10.1021/acsami.1c22280

Figure Lengend Snippet: Recombinant human lubricin and lubricin subjected to accelerated aging conditions adsorbed to StcE-treated differentiated hTCEpi cells in a dose-dependent manner (scale bar: 100 μm).

Article Snippet: Recombinant human lubricin was generously provided by Novartis (Basel, Switzerland) at a concentration of 2.01 mg/mL (including O -glycosylation) in 10 mM sodium phosphate, 140 mM sodium chloride, and 0.02% (w/v) polysorbate 20, pH 7.0.

Techniques: Recombinant

Journal: ACS Applied Materials & Interfaces

Article Title: A Mucin-Deficient Ocular Surface Mimetic Platform for Interrogating Drug Effects on Biolubrication, Antiadhesion Properties, and Barrier Functionality

doi: 10.1021/acsami.1c22280

Figure Lengend Snippet: Lubricin supplementation reduced rose bengal penetrance in a dose-dependent manner for both unstressed and aged lubricin on differentiated hTCEpi cells. (A) StcE-induced mucin deficiency increased dye penetrance, which was attenuated by recombinant human lubricin in a dose-dependent manner. (B–E) Supplementation with higher concentrations of (B) lubricin or lubricin subjected to (C) temperature stress, (D) acid stress, or (E) alkaline stress resulted in an increased rose bengal-negative area. (F) Trends in the excluded area for different lubricin variants were consistent across three concentrations (25, 75, and 250 μg/mL). Statistical significance ( p < 0.05) was determined using one-way Welch's ANOVA with post-hoc Dunnett’s T3 multiple comparisons testing relative to StcE-treated (0 μg/mL) cells (B–E) or unstressed drug substance (DS) (F).

Article Snippet: Recombinant human lubricin was generously provided by Novartis (Basel, Switzerland) at a concentration of 2.01 mg/mL (including O -glycosylation) in 10 mM sodium phosphate, 140 mM sodium chloride, and 0.02% (w/v) polysorbate 20, pH 7.0.

Techniques: Recombinant