anti integrin α v β 5  (Santa Cruz Biotechnology)

Journal: iScience

Article Title: Anti-angiogenic collagen IV-derived peptide target engagement with α v β 3 and α 5 β 1 in ocular neovascularization models

doi: 10.1016/j.isci.2023.106078

Figure Lengend Snippet:

Article Snippet: C overnight with rabbit antibodies directed against α v β 3 (1:100, Abbiotec, Escondido, CA; Cat # 251672), α 5 β 1 (1:100, Novus Biological, Centennial, CO; Cat # nbp2-29788), α 5 (1:200, Millipore Sigma, Burlington, MA; Cat # AB1928), α v (1:200, Millipore Sigma, Burlington, MA; Cat # AB1930) or AXT107 (1:100, Asclepix, Baltimore, MD) in blocking solution.

Techniques: Recombinant, Protease Inhibitor, Electron Microscopy, Plasmid Preparation, Transgenic Assay, Software, Diagnostic Assay, Microinjection

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

Journal: Frontiers in ophthalmology

Article Title: miR-92a and integrin expression in fibrovascular membranes in proliferative diabetic retinopathy

doi: 10.3389/fopht.2023.1116838

Figure Lengend Snippet: Increase in an integrin α v β 3 and α 5 expressions in FVM of PDR subjects. Representative photomicrographs showing staining and bar chart quantifying fluorescence intensity for α v β 3 (A, B) and α 5 (C, D) integrins in the FVM membranes of PDR subjects and control (macular pucker) individuals n=9 (3 images X 3 independent biological replicates). *p<0.05, **p<0.01.

Article Snippet: The sections of 8 μm thickness were obtained using Leica CM3050 Cryostat (Buffalo Grove, IL) followed by staining using integrin α 5 (1:100, #CBL497, Millipore Burlington, MA) or α v β 3 antibodies, (1:100, #MAB1976 Millipore).

Techniques: Staining, Fluorescence, Control

Journal: iScience

Article Title: Anti-angiogenic collagen IV-derived peptide target engagement with α v β 3 and α 5 β 1 in ocular neovascularization models

doi: 10.1016/j.isci.2023.106078

Figure Lengend Snippet: Integrin monomers α v and α 5 are detected in blood vessels in normal retina and in neovascularization in ischemic retina At P17, ocular frozen sections from normal C57BL/6 mice (n = 10, A–D and I–L) or from mice with oxygen-induced retinopathy (n = 10, E–H and M−P) were stained with Alexa Fluor 594-conjugated Griffonia Simplicifolia Agglutinin (GSA) lectin that selectively stains vascular cells and immunostained for αv and α5. A minimum of 20 ocular sections were assessed per group. The study was replicated, and it showed qualitatively similar result each time. Scale bar = 100 μm.

Article Snippet: Immunofluorescent staining was also performed on cultured vascular endothelial cells using primary antibodies directed against integrin α 5 (Millipore, Burlington, MA; Cat # AB1928) or integrin α v (Millipore, Burlington, MA; Cat # AB1930).

Techniques: Staining

Journal: iScience

Article Title: Anti-angiogenic collagen IV-derived peptide target engagement with α v β 3 and α 5 β 1 in ocular neovascularization models

doi: 10.1016/j.isci.2023.106078

Figure Lengend Snippet: Integrins α v β 3 and α 5 β 1 are upregulated in neovascularization and some pre-existent retinal vessels in ocular sections of ischemic retina At P17, C57BL/6 room air control mice (n = 10) or mice with oxygen-induced ischemic retinopathy (n = 10, OIR) were euthanized and ocular sections were stained with Alexa Fluor 594-conjugated Griffonia Simplicifolia Agglutinin (GSA) lectin and immunostained for α v β 3 or α 5 β 1 . There was no α v β 3 detectable on GSA-labeled retinal vessels in room air control retina (A–C), but it was detectable in NV and pre-existent vessels in ischemic retina ocular sections from P17 mice with OIR (D–F). Similarly, integrin α 5 β 1 was not detectable on GSA-labeled retinal vessels in room air control retina (G–I), but it was detectable in NV and pre-existent vessels in ocular sections (J–L) from mice with OIR. A minimum of 20 ocular sections were assessed per group. The study was replicated, and it showed qualitatively similar result each time. Scale bar = 100 μm.

Article Snippet: Immunofluorescent staining was also performed on cultured vascular endothelial cells using primary antibodies directed against integrin α 5 (Millipore, Burlington, MA; Cat # AB1928) or integrin α v (Millipore, Burlington, MA; Cat # AB1930).

Techniques: Control, Staining, Labeling

Journal: iScience

Article Title: Anti-angiogenic collagen IV-derived peptide target engagement with α v β 3 and α 5 β 1 in ocular neovascularization models

doi: 10.1016/j.isci.2023.106078

Figure Lengend Snippet: Integrins α v β 3 and α 5 β 1 are upregulated in neovascularization in flat mounts of ischemic retina At P17, C57BL/6 room air control mice (n = 10) or mice with oxygen-induced ischemic retinopathy (n = 10, OIR) were euthanized and retinal flat mounts were stained with Alexa Fluor 594-conjugated Griffonia Simplicifolia Agglutinin (GSA) lectin and immunostained for α v β 3 or α 5 β 1 . There was no α v β 3 detectable on GSA-labeled retinal vessels in room air control retina (A–C), but it was detectable in buds of NV on retinal flat mounts from P17 mice with OIR (D–F). Similarly, integrin α 5 β 1 was not detectable on GSA-labeled retinal vessels in room air control retina (G–I), but it was detectable in NV in retinal flat mounts (J–L) from mice with OIR. The experiment was performed in duplicates, and qualitatively similar results were found in the replicates. Scale bar = 100 μm.

Article Snippet: Immunofluorescent staining was also performed on cultured vascular endothelial cells using primary antibodies directed against integrin α 5 (Millipore, Burlington, MA; Cat # AB1928) or integrin α v (Millipore, Burlington, MA; Cat # AB1930).

Techniques: Control, Staining, Labeling

Journal: iScience

Article Title: Anti-angiogenic collagen IV-derived peptide target engagement with α v β 3 and α 5 β 1 in ocular neovascularization models

doi: 10.1016/j.isci.2023.106078

Figure Lengend Snippet: AXT107 cross-links with integrins α 5 and β 3 P16 mice reared in room air (1 set of 7 mice) or OIR (2 sets of 7 mice) were injected intravitreally with sulfo-SBED-labeled AXT107, and their retinas were isolated after 24h. Bound peptide was then cross-linked using UV reactive chemistry, and the tissue was lysed. Each lysate was then separated into three types of samples called input, pull-down, or flowthrough for the lysates before pull-down, components that associated with the beads, and components that did not associate with the beads, respectively. (A–D) Input samples from each treatment set were separated by SDS-PAGE and analyzed by western blot for the levels of integrins α 5 (A), α v (B), β 1 (C), and β 3 (D) with β-tubulin as a loading control. (E–G) Input, pull-down, and flowthrough samples from each of the three datasets resolved by SDS-PAGE and analyzed for integrins α 5 (E), α v (F), and β 1 and β 3 (G) with β-tubulin as a loading control. Input samples for integrin α 5 are an extended cropping of the gel used for panel A. Conversely, the input samples in panels F and G are included to allow same gel comparisons of the three different samples but were rerun for panels B, C, and D to address an overloading artifact in lane 1. Owing to differences in molecular weights and staining intensity, integrins β 1 and β 3 were stained serially on the same blot.

Article Snippet: Immunofluorescent staining was also performed on cultured vascular endothelial cells using primary antibodies directed against integrin α 5 (Millipore, Burlington, MA; Cat # AB1928) or integrin α v (Millipore, Burlington, MA; Cat # AB1930).

Techniques: Injection, Labeling, Isolation, SDS Page, Western Blot, Control, Staining

Journal: iScience

Article Title: Anti-angiogenic collagen IV-derived peptide target engagement with α v β 3 and α 5 β 1 in ocular neovascularization models

doi: 10.1016/j.isci.2023.106078

Figure Lengend Snippet:

Article Snippet: Immunofluorescent staining was also performed on cultured vascular endothelial cells using primary antibodies directed against integrin α 5 (Millipore, Burlington, MA; Cat # AB1928) or integrin α v (Millipore, Burlington, MA; Cat # AB1930).

Techniques: Recombinant, Protease Inhibitor, Electron Microscopy, Plasmid Preparation, Transgenic Assay, Software, Diagnostic Assay, Microinjection