Journal: Cell Communication and Signaling : CCS

Article Title: Endomucin selectively regulates vascular endothelial growth factor receptor-2 endocytosis through its interaction with AP2

doi: 10.1186/s12964-024-01606-w

Figure Lengend Snippet: Essential role of EMCN in facilitating VEGFR2 and AP2 interaction and clathrin recruitment. A Colocalization of clathrin HC (heavy chain) (green) and VEGFR2 (red) were visualized in control HRECs with or without VEGF165 (10 ng/ml). Examples of colocalization of VEGFR2 and clathrin HC (white arrowhead), and clathrin (white arrow) were shown in magnified view. Bar = 10 µm B Fraction of VEGFR2 that colocalized with clathrin was quantified by Image J CoJAP plugin. Student t-test was used for the comparison. * P < 0.05, n = 3. C Colocalization of clathrin HC (heavy chain) (green) and VEGFR2 (red) were visualized in siNT or siEMCN HRECs with VEGF165 (10 ng/ml) stimulation. Examples of colocalization of VEGFR2 and clathrin HC (white arrowheads), and VEGFR2 (white arrow) were shown in magnified view. Bar = 10 µm D Fraction of VEGFR2 that colocalized with clathrin in siNT and siEMCN HRECs was quantified. Student t-test was used for the comparison. ** P < 0.01, n = 3. E Colocalization of clathrin HC (heavy chain) (green) and AP2 (red) were visualized in control in siNT and siEMCN HRECs with VEGF(10 ng/ml) stimulation. Examples of colocalization of clathrin HC and AP2 (white arrowhead) were shown in magnified view. Bar = 10 µm ( F ) Fraction of clathrin that colocalized with AP2 in siNT and siEMCN HRECs with VEGF stimulation were quantified. Student t-test was used for the comparison. P > 0.05, n = 3. G EMCN is required for interaction between VEGFR2 and AP2A2. HRECs were lysed and VEGFR2 that co-immunoprecipitated with AP2A2 in the presence and absence of EMCN was observed. n = 3

Article Snippet: Recombinant human VEGF165 (#293-VE), PlGF-2 (#6837-PL), and FGF2 (#233-FB) were purchased from R&D Systems.

Techniques: Control, Comparison, Immunoprecipitation

Journal: Cell Communication and Signaling : CCS

Article Title: Endomucin selectively regulates vascular endothelial growth factor receptor-2 endocytosis through its interaction with AP2

doi: 10.1186/s12964-024-01606-w

Figure Lengend Snippet: EMCN does not modulate VEGF165 or PIGF-induced endothelial migration or VEGFR1 internalization. A EMCN knockdown did not affect PlGF-2-induced HREC migration. HRECs were transfected with either siNT or siEMCN, mechanically scratched, stimulated with PlGF-2 (10 ng/ml) or VEGF165 (10 ng/ml), and the resulting cell migration was quantified by image analysis (left). ** P < 0.01, n = 6 or 9. Representative images of each group at time zero (white dashed line) and 15 h time (yellow dashed line) points (right). The scale bar represents 500 µm. B Illustration of the cell surface receptor internalization assay. Growth factors bind to its cell surface receptors and induce receptor internalization. Cell surface proteins are biotinylated, the cell surface fraction is separated using avidin resin, and western blot analysis were used to analyze the fraction of receptors remaining at the cell surface. C HRECs incubated in serum-free media were stimulated with VEGF165 (10 ng/ml) for 30 min with and without EMCN knockdown, and cell surface membrane-bound VEGFR1 (mVEGFR1) levels were analyzed by western blot analysis. * P < 0.05, ** P < 0.01, *** P < 0.001, n = 6. One-way ANOVA was used for statistical analysis

Article Snippet: Recombinant human VEGF165 (#293-VE), PlGF-2 (#6837-PL), and FGF2 (#233-FB) were purchased from R&D Systems.

Techniques: Migration, Knockdown, Transfection, Cell Surface Receptor Assay, Avidin-Biotin Assay, Western Blot, Incubation, Membrane

Journal: Cell Communication and Signaling : CCS

Article Title: Endomucin selectively regulates vascular endothelial growth factor receptor-2 endocytosis through its interaction with AP2

doi: 10.1186/s12964-024-01606-w

Figure Lengend Snippet: EMCN is not required for FGF2-induced HREC cell migration or FGFR1 internalization. A EMCN knockdown did not affect FGF2-induced HREC migration. HRECs were transfected with either siNT or siEMCN, incubated in serum-free media for 8 h, mechanically scratched, and stimulated with FGF2 (10 ng/ml) or VEGF165 (10 ng/ml). Quantification of cell migration for all the treatment groups based on image analysis (left). Student t-test was used for comparisons within groups. * P < 0.05, n = 8. Representative images of each group at time zero (white dashed line) and 15 h time (yellow dashed line) points (right). The scale bar represents 500 µm. B EMCN knockdown did not affect FGF2-induced FGFR1 internalization in HREC. Serum-starved HRECs were stimulated with FGF2 for 45 min, and then the cell surface proteins were isolated and visualized by western blot. Quantification of FGFR1 at the cell surface from all treatment groups by western blot analysis (left). Student-t test was used for statistical analysis. * P < 0.05, n = 7. A representative western blot for all treatment groups (right). C EMCN does not interact with VEGFR1 or FGFR1 in HRECs. HRECs overexpressing myc-tagged EMCN were lysed, and cell surface receptors that co-immunoprecipitated with EMCN were observed. n = 3. Note that the IgG and Myc groups were overexposed together for the better detection of the different receptors because of the low protein levels, while the input groups were kept at a lower exposure

Article Snippet: Recombinant human VEGF165 (#293-VE), PlGF-2 (#6837-PL), and FGF2 (#233-FB) were purchased from R&D Systems.

Techniques: Migration, Knockdown, Transfection, Incubation, Isolation, Western Blot, Immunoprecipitation

Journal: Cell Communication and Signaling : CCS

Article Title: Endomucin selectively regulates vascular endothelial growth factor receptor-2 endocytosis through its interaction with AP2

doi: 10.1186/s12964-024-01606-w

Figure Lengend Snippet: EMCN knockdown inhibits VEGF121 induced VEGFR2 internalization and HRECs migration similar to VEGF165. A Both VEGF165 (10 ng/ml)- and VEGF121 (7.29 ng/ml)-induced migration were inhibited with EMCN knockdown. Quantification of cell migration by image analysis is shown (left). Student-t test was used for comparisons between groups. * P < 0.05, ** P < 0.01, n = 10. Representative images of each group at time zero (white dashed line) and 15 h time (yellow dashed line) points. The scale bar represents 500 µm. B Schematic representation of VEGFA isoforms, VEGF165 and VEGF121. C HRECs were treated with siEMCN stimulated with VEGF121 (7.29 ng/ml) for a time course of up to 120 min. VEGF121 induced significant VEGFR2 endocytosis after 60 min of stimulation, except when EMCN was knockdown. One-way ANOVA was used for comparation within group. Student-t test was used for comparation between siNT and siEMCN at the same time point. # P < 0.05, * P < 0.05, *** P < 0.001, **** P < 0.0001, n = 3. D Representative image of the western blot for VEGFR2 internalization in both siNT and siEMCN groups. CD31 was blotted as cell surface fraction loading control

Article Snippet: Recombinant human VEGF165 (#293-VE), PlGF-2 (#6837-PL), and FGF2 (#233-FB) were purchased from R&D Systems.

Techniques: Knockdown, Migration, Western Blot, Control

Journal: Neural Regeneration Research

Article Title: Telencephalic stab wound injury induces regenerative angiogenesis and neurogenesis in zebrafish: unveiling the role of vascular endothelial growth factor signaling and microglia

doi: 10.4103/NRR.NRR-D-23-01881

Figure Lengend Snippet: Analysis of regenerative angiogenesis and neurogenesis after VEGF165 injection. (A) PCNA immunohistostaining (proliferating cells in purple) in telencephalon sections of Tg( fli:EGFP × mpeg1.1:mCherry ) zebrafish (blood vessels in green) after microinjection with PBS (vehicle) and VEGF165 (50 µg/mL) at the SW injury site. Scale bars: 200 µm. (B–D) Quantification of different angiogenic parameters shows an increasing regenerative angiogenesis tendency in the SW hemisphere of VEGF165-injected zebrafish compared with the SW hemisphere of PBS-injected zebrafish at 1 dpl. (E) Quantification of the ventricular proliferative surface of fish injected with PBS and VEGF165 showing an increasing tendency for NSC proliferation. The data are presented as the mean ± standard error of the mean ( n = 5–6 per condition from two independent experiments) and were analyzed with Student’s t -test. * P < 0.05, vs . the SW hemisphere of PBS-injected zebrafish. dpl: Day(s) post-lesion; EGFP: enhanced green fluorescent protein; GFP: green fluorescent protein; PBS: phosphate-buffered saline; PCNA: proliferating cell nuclear antigen; SW: stab-wounded; VEGF: vascular endothelial growth factor.

Article Snippet: The positive effects of VEGF on angiogenesis and brain regeneration were investigated by injecting recombinant zebrafish VEGF165 at the lesion site: the fish received an intraparenchymal microinjection with 1× PBS or 50 μg/mL VEGF165 in 1× PBS (REF: 1247-ZV/CF, R&D Systems, Noyal Châtillon sur Seiche, France).

Techniques: Injection, Microinjection, Saline

Journal: Neural Regeneration Research

Article Title: Telencephalic stab wound injury induces regenerative angiogenesis and neurogenesis in zebrafish: unveiling the role of vascular endothelial growth factor signaling and microglia

doi: 10.4103/NRR.NRR-D-23-01881

Figure Lengend Snippet: Microglial recruitment is regulated by Vegf signaling. The SW hemisphere was compared with the respective contralateral hemisphere at 3 dpl. (A, C) Telencephalon sections of Tg( fli:EGFP × mpeg1.1:mCherry ) zebrafish (microglia in red). Scale bars: 200 µm. (B) Quantification of mpeg + cells showing a decrease in tivozanib-injected zebrafish compared with DMSO-injected zebrafish. (D) Quantification of mpeg + cells showing an increasing tendency in VEGF165-injected zebrafish compared with PBS-injected zebrafish. The data are presented as the mean ± standard error of the mean ( n = 5–6 per condition from two independent experiments) and were analyzed with Student’s t -test. *** P < 0.001, vs . the contralateral hemisphere. DMSO: Dimethyl sulfoxide; dpl: day(s) post-lesion; mpeg: macrophage‐expressed gene; PBS: phosphate-buffered saline; SW: stab-wounded; Vegf/VEGF: vascular endothelial growth factor.

Article Snippet: The positive effects of VEGF on angiogenesis and brain regeneration were investigated by injecting recombinant zebrafish VEGF165 at the lesion site: the fish received an intraparenchymal microinjection with 1× PBS or 50 μg/mL VEGF165 in 1× PBS (REF: 1247-ZV/CF, R&D Systems, Noyal Châtillon sur Seiche, France).

Techniques: Injection, Saline

Journal: Neural Regeneration Research

Article Title: Telencephalic stab wound injury induces regenerative angiogenesis and neurogenesis in zebrafish: unveiling the role of vascular endothelial growth factor signaling and microglia

doi: 10.4103/NRR.NRR-D-23-01881

Figure Lengend Snippet: Microglial and endothelial cell proliferation following brain lesion and modulation of Vegf signaling. (A) Representative images of PCNA immunostaining of Tg( fli:EGFP × mpeg1.1:mCherry ) zebrafish following microlesion/injection with DAPI counterstaining. (B) Higher magnification of the white panels in A illustrating endothelial cell proliferation (Fli + /PCNA + ) and microglial proliferation (mpeg + /PCNA + ). Scale bar: 120 µm (A) and 70 µm and magnified 7 µm (B). (C–E) Quantification of PCNA + , PCNA + /Fli + , and PCNA + /mpeg + cells in the SW hemisphere following phosphate-buffered saline (PBS) and VEGF165 microinjection. (F–H) Quantification of PCNA + , PCNA + /Fli + , and PCNA + /mpeg + cells in the SW hemisphere following DMSO and tivozanib microinjection. The data are presented as the mean ± standard error of the mean ( n = 5–6 per condition from two independent experiments) and were analyzed with Student’s t -test. * P < 0.05, vs . the lesioned hemisphere of the respective control. DAPI: 4′,6-Diamidino-2-phenylindole; DMSO: dimethyl sulfoxide; EGFP: enhanced green fluorescent protein; mpeg: macrophage‐expressed gene; PBS: phosphate-buffered saline; PCNA: proliferating cell nuclear antigen; SW: stab-wounded; Vegf/VEGF: vascular endothelial growth factor.

Article Snippet: The positive effects of VEGF on angiogenesis and brain regeneration were investigated by injecting recombinant zebrafish VEGF165 at the lesion site: the fish received an intraparenchymal microinjection with 1× PBS or 50 μg/mL VEGF165 in 1× PBS (REF: 1247-ZV/CF, R&D Systems, Noyal Châtillon sur Seiche, France).

Techniques: Immunostaining, Injection, Saline, Microinjection, Control