α v β 5 integrin  (MedChemExpress)

Journal: Science Advances

Article Title: Integrin inhibition facilitates fibrocartilaginous transformation in connective tissue in osteoarthritis

doi: 10.1126/sciadv.ady4112

Figure Lengend Snippet: ( A ) Dot plot shows notably increased ECM-related signaling pathways of FB4 ( COL5A1 + ) in ADD TMJ-OA RCT compared to normal RCT. ( B ) GO analysis shows potentially up-regulated gene functions of FB4 ( COL5A1 + ) in ADD TMJ-OA RCT compared to normal RCT. Top 15 GO terms of the enrichment factors are showed. MHC, major histocompatibility complex; ER, endoplasmic reticulum. ( C ) Dot plot shows the expression of integrin-related genes in FB4 ( COL5A1 + ). The red (blue) dots are the up-regulated (down-regulated) genes in ADD TMJ-OA RCT compared to normal RCT. Horizontal and vertical coordinates indicate the specificity of the α and β integrin subunit genes on FB4 ( COL5A1 + ) cells. ( D ) The chord diagram shows the ligand-receptor pairs of periostin signaling (left). Proportions of periostin signaling interaction strength are compared between normal RCT and ADD TMJ-OA RCT (right). ( E ) Left plot shows coexpression of ITGAV and ITGB5 in FB4 ( COL5A1 + ) cells on the 10x Visium capture slides. Proportions of the coexpression of ITGAV and ITGB5 in FB4 ( COL5A1 + ) cells are compared between normal RCT and ADD TMJ-OA RCT. ( F ) Immunofluorescent staining for integrin α V β 5 in human TMJ disk tissues. Red: integrin α V β 5 ; blue: DAPI. White dotted lines: boundary of the disk tissues. N = 3 independent samples from different donors. Scale bars, 500 μm (left) and 50 μm (right). ( G ) Semiquantification of the percentage of integrin α V β 5 + area. Data are presented as mean ± SD. N = 3 independent animals. The two-tailed t test is used for data analysis. ** P < 0.01.

Article Snippet: Primary antibodies, which included antibodies against integrin α 5 β V (MAB2528, AB_2280706, R&D Systems, USA) diluted to 1:100 in blocking solution, were incubated at 4°C overnight.

Techniques: Protein-Protein interactions, Immunopeptidomics, Expressing, Staining, Two Tailed Test

Journal: Molecular Therapy

Article Title: Pharmacological blocking of microfibrillar-associated protein 4 reduces retinal neoangiogenesis and vascular leakage

doi: 10.1016/j.ymthe.2025.01.038

Figure Lengend Snippet: MFAP4 binding and activation of RGD-dependent integrins (A) MFAP4 promotes adhesion of human pulmonary microvascular endothelial cells (HPMECs) in a dose-dependent manner. (B) MFAP4-mediated adhesion of HPMEC can be inhibited by RGD-containing peptide (GRGDS) but not control (SDGRG) peptide. Data are means (SD) of n = 3 independent experiments. Significance is calculated by two-way ANOVA. MFI, mean fluorescence intensity. Immobilized recombinant MFAP4 (2 μg/mL) was incubated with increasing concentrations of recombinant integrins. Integrin binding to recombinant MFAP4 was detected for (C) integrins α V β 3/5/6 and αIIbβ 3 but not (D) integrins α V β 1/8 and α 4/5/8 β 1 . Used detection antibodies are shown in brackets. Data are shown as means (SD) of n = 3 independent experiments. Relative band density of (E) phosphorylated FAK (pFAK)/total FAK and (F) pERK/total ERK in HPMEC after cellular adhesion to poly-D-lysine (PDL, negative control), vitronectin (VTN, positive control), or MFAP4 are shown in representative western blots and quantitated mean (SD) of n = 3 independent experiments. Quantifications of western blotting was analyzed by two-way ANOVA for MFAP4 relative to negative control and the significance is provided for treatment factor only (independent of the significant time factor).

Article Snippet: HPMECs were detached with Accutase (Fisher Scientific) and suspended at 200,000 cells/sample in FACS buffer containing 10 μg/mL mouse mAbs against integrin α V β 3 (Millipore, cat. # MAB1976), integrin α V β 5 (Santa Cruz, cat. # sc-13588), with anti-ovalbumin (HYB099-01), The State Serum Institute) as isotype control (IC) for 1 h at 4°C.

Techniques: Binding Assay, Activation Assay, Control, Fluorescence, Recombinant, Incubation, Negative Control, Positive Control, Western Blot

Journal: Molecular Therapy

Article Title: Pharmacological blocking of microfibrillar-associated protein 4 reduces retinal neoangiogenesis and vascular leakage

doi: 10.1016/j.ymthe.2025.01.038

Figure Lengend Snippet: The AS0326 antibody blocks MFAP4s interaction with endothelial integrins (A) HPMECs were subjected to MFAP4-mediated adhesion, and inhibition of adhesion was tested with integrin α V β 3 - or integrin α V β 5 -blocking antibodies. (B) mAS0326 antibody (mAS) blocks HPMEC adhesion to MFAP4. MFI is of fluorescently labeled cells. IC, isotype control. Significance is calculated relative to the MFAP4-treated positive control in (A) and (B). Flow cytometry staining of (C) integrin α V β 3 and (D) integrin α V β 5 in HPMEC compared to IC. Representative histograms of n = 3 independent experiments are shown. Human primary retinal endothelial cells (RECs) were seeded on immobilized albumin or MFAP4. RECs were stimulated with VEGF and 24-h proliferation was assessed. (E) REC proliferation was co-treated with integrin α V β 3 - or α V β 5 -blocking antibodies or hAS0326 (hAS). REC migration for 3.5 h on MFAP4-coated surface was assessed by Transwell assay using VEGF as chemoattractant. (F) MFAP4-dependent migration was treated with integrin α V β 3 - or α V β 5 -blocking antibodies or hAS0326. Data are shown as individual datapoints with mean (SD) of n = 3 independent experiments. Data are normalized to albumin control. Significance is calculated relative to the hAS0326 treatment group for (E) and (F). Significance calculations are performed using one-way ANOVA followed by Dunnett’s multiple comparison test. Representative images of REC’s (purple) migrated through the pores of the Transwell assay inserts when migration on (G) albumin-coated insert or (H) MFAP4-coated insert. Bar, 100 μm. All antibodies were provided in 10 μg/mL doses for (A), (B), (E), and (F).

Article Snippet: HPMECs were detached with Accutase (Fisher Scientific) and suspended at 200,000 cells/sample in FACS buffer containing 10 μg/mL mouse mAbs against integrin α V β 3 (Millipore, cat. # MAB1976), integrin α V β 5 (Santa Cruz, cat. # sc-13588), with anti-ovalbumin (HYB099-01), The State Serum Institute) as isotype control (IC) for 1 h at 4°C.

Techniques: Inhibition, Blocking Assay, Labeling, Control, Positive Control, Flow Cytometry, Staining, Migration, Transwell Assay, Comparison

Journal: Molecular Therapy

Article Title: Pharmacological blocking of microfibrillar-associated protein 4 reduces retinal neoangiogenesis and vascular leakage

doi: 10.1016/j.ymthe.2025.01.038

Figure Lengend Snippet: Specificity of the AS0326 antibody (A) mAS0326 and (B) hAS0326 efficiently detect MFAP4 in WT ( Mfap4 +/+ ) mouse serum, but not in MFAP4-deficient ( Mfap4 −/− ) mouse serum. (C) Increasing concentrations of hAS0326-Fab or hAS0326Y94A L-CDR3 Fab variant were applied for inhibition of 1.5 nM MFAP4 binding to an excess of immobilized integrin α V β 3 . (D–F) Brown symbols, HG-HYB7-5; purple symbols, mAS0326; red symbols; hAS0326. (D) Both mAS0326 and hAS0326 efficiently detect immobilized recombinant human MFAP4 (rhMFAP4 coating) variant carrying RGD-AAA mutation (RGD-AAA), while HG-HYB7-5 antibody show RGD-dependent binding. Both mAS0326 and hAS0326 efficiently detect immobilized (E) recombinant mouse (rm) MFAP4, and (F) rhMFAP4, while HG-HYB7-5 is rhMFAP4 specific. Data are shown as means (SD) of n = 3 independent experiments.

Article Snippet: HPMECs were detached with Accutase (Fisher Scientific) and suspended at 200,000 cells/sample in FACS buffer containing 10 μg/mL mouse mAbs against integrin α V β 3 (Millipore, cat. # MAB1976), integrin α V β 5 (Santa Cruz, cat. # sc-13588), with anti-ovalbumin (HYB099-01), The State Serum Institute) as isotype control (IC) for 1 h at 4°C.

Techniques: Variant Assay, Inhibition, Binding Assay, Recombinant, Mutagenesis

Journal: Molecular Therapy

Article Title: Pharmacological blocking of microfibrillar-associated protein 4 reduces retinal neoangiogenesis and vascular leakage

doi: 10.1016/j.ymthe.2025.01.038

Figure Lengend Snippet: Structural basis for the hAS0326 Fab interaction with MFAP4 The hAS0326 Fab complex and the MFAP4 octamer. (A) Cartoon representation of the octameric MFAP4-Fab complex. (B) Each Fab contacts a single MFAP4 monomer at an epitope next to the MFAP4 N terminus but far from the binding site for the Ca 2+ ion (cyan sphere). (C) The MFAP4 octamer is built from two FIBCD1-like tetramers (colored orange and red) with eight Ca 2+ ions located at the tetramer-tetramer interface. (D) Inside view showing the concave face of the tetramer. (E) Top view of the convex face of the tetramer with the four bound Fab molecules. Orange spheres mark the first MFAP4 residue that can be located. Presumably, the disordered RGD integrin-binding motif at the N terminus of MFAP4 is trapped and inaccessible inside the funnel-shaped space formed by the Fab molecules. Nt, N terminus. (F) The epitope mapped on MFAP4. Residues primarily in contact with heavy-chain and light-chain CDRs are colored blue and green, respectively. (G) Details of the intermolecular interaction centered on MFAP4 residues 24–29. Putative polar interactions are indicated by dotted lines.

Article Snippet: HPMECs were detached with Accutase (Fisher Scientific) and suspended at 200,000 cells/sample in FACS buffer containing 10 μg/mL mouse mAbs against integrin α V β 3 (Millipore, cat. # MAB1976), integrin α V β 5 (Santa Cruz, cat. # sc-13588), with anti-ovalbumin (HYB099-01), The State Serum Institute) as isotype control (IC) for 1 h at 4°C.

Techniques: Binding Assay, Residue

Journal: Molecular Therapy

Article Title: Pharmacological blocking of microfibrillar-associated protein 4 reduces retinal neoangiogenesis and vascular leakage

doi: 10.1016/j.ymthe.2025.01.038

Figure Lengend Snippet: The macular proteome of the DL-AAA-induced model of chronic retinopathy supports integrin involvement Macular punches including choroid, RPE, and retina obtained at end-study (week 22) were analyzed by mass spectrometry and following analysis using clusterProfiler 4.0 package in R. The proteomes were generated from n = 3 eyes for non-diseased control, n = 5 eyes for DL-AAA treatment, and n = 5 eyes for hAS0326 treatment. (A) Over-representation analysis (ORA) showing the effect of DL-AAA treatment (relative to no DL-AAA treatment) and hAS0326 treatment of DL-AAA-treated eyes (relative to DL-AAA treatment), respectively. The plot was generated using compareCluster function with default settings. All ontologies with significant regulation post correction for multiple testing using the Benjamini-Hochberg procedure are shown. Dot sizes indicate the ratio (i.e., the coverage of a given term by proteins regulated for each comparison), and dot colors indicate the level of significance. (B–E) Volcano plots showing regulation of detected proteins underlying selected GO terms. For selected, significantly regulated gene ontologies, the ORA input proteins displaying significant regulation are highlighted in color. Protein IDs are shown for the top three proteins with lowest p belonging to a particular ontology. Moreover, protein IDs are shown for specific proteins of interest.

Article Snippet: HPMECs were detached with Accutase (Fisher Scientific) and suspended at 200,000 cells/sample in FACS buffer containing 10 μg/mL mouse mAbs against integrin α V β 3 (Millipore, cat. # MAB1976), integrin α V β 5 (Santa Cruz, cat. # sc-13588), with anti-ovalbumin (HYB099-01), The State Serum Institute) as isotype control (IC) for 1 h at 4°C.

Techniques: Mass Spectrometry, Generated, Control, Comparison