Journal: Frontiers in Neurology

Article Title: Repulsive Guidance Molecule a Inhibits Angiogenesis by Downregulating VEGF and Phosphorylated Focal Adhesion Kinase In Vitro

doi: 10.3389/fneur.2017.00504

Figure Lengend Snippet: Repulsive guidance molecule a (RGMa) expression increased in human umbilical artery endothelial cells (HUAECs), human umbilical vein endothelial cells (HUVECs), and rat brain microvascular endothelial cells (RBMECs) stimulated with VEGF. (A,B) Endothelial cell (EC) migration distance was evaluated by scratch assay. ECs were treated with a dose curve of RGMa (500–3000 ng/ml), and images were taken at the beginning and 12 h. (C,D) ECs were treated with RGMa (2 µg/ml), and lysates were collected over a time course of 50 min. The amount of phosphorylated focal adhesion kinase (p-FAK) protein was visualized with western blot analysis. (E,F) ECs were treated with VEGF (50 ng/ml), and lysates were collected over a course of 120 min. p-FAK protein levels were visualized with western blot analysis. (G–I) Quantitative real-time polymerase chain reaction showed RGMa mRNA level was upregulated in HUAECs, HUVECs, and RBMECs exposed to VEGF (50 ng/ml) for 30 min. (J–L) RGMa protein levels were visualized in HUAECs, HUVECs, and RBMECs exposed to VEGF at 30 min by western blot analysis. Data in bar graphs represent the means ± SD of ≥4 independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001, # VS 1500 ng/ml, ### P < 0.001.

Article Snippet: Recombinant human RGMa, recombinant rat RGMa, recombinant human VEGF, and recombinant rat VEGF were obtained from R&D Systems (MN, USA).

Techniques: Expressing, Migration, Wound Healing Assay, Western Blot, Real-time Polymerase Chain Reaction

Journal: Frontiers in Neurology

Article Title: Repulsive Guidance Molecule a Inhibits Angiogenesis by Downregulating VEGF and Phosphorylated Focal Adhesion Kinase In Vitro

doi: 10.3389/fneur.2017.00504

Figure Lengend Snippet: Repulsive guidance molecule a (RGMa) suppressed VEGF expression, phosphorylation of focal adhesion kinase (FAK), proliferation, migration, and tube formation in ECs. (A,B) Lysate was collected, and VEGF was detected by western blot in human umbilical artery endothelial cells (HUAECs) treated with RGMa (2 µg/ml); (C–E) ELISA kit assay showed VEGFA decreased in endothelial cell (EC)-culture supernatant exposed to RGMa compared with cell-culture supernatant from control group. (F–H) Cell proliferation was evaluated with cell-counting kit-8 and 5-ethynyl-2′-deoxyuridine (EdU) assays (I,J) . RGMa decreased proliferation of ECs stimulated and unstimulated with VEGF. (K,L) FAK (Tyr397) phosphorylation was measured with western blot in HUAECs treated with vehicle, RGMa (2 µg/ml), VEGF (50 ng/ml), or VEGF plus RGMa. (M–P) ECs were grown to 100% confluence, serum-starved overnight, wounded with a sterile pipette tip to remove cells, and treated with control, RGMa, VEGF, or VEGF plus RGMa. Photographs (40×) were taken at 12 h after injury. Wound closure of ≥3 wells was quantified and reported as mean ± SD. (Q–T) Migration activity of ECs treated with RGMa, VEGF, VEGF plus RGMa, or control was measured with transwell assay. Photographs (200×) were taken 18 h after treatment. (U–X) HUAECs were starved overnight, treated as indicated, and seeded into 96-well plates coated with Matrigel. Photographs (40×) were taken at 3 h after treatment. The number of tubes, tube area, and tube length were analyzed with Image J. Scale bar, 100 µm. Data shown are representative of experimental and quantitative results. N ≥ 4 independent experiments. Bars represent mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Recombinant human RGMa, recombinant rat RGMa, recombinant human VEGF, and recombinant rat VEGF were obtained from R&D Systems (MN, USA).

Techniques: Expressing, Phospho-proteomics, Migration, Western Blot, Enzyme-linked Immunosorbent Assay, Cell Culture, Control, Cell Counting, Sterility, Transferring, Activity Assay, Transwell Assay

Journal: Frontiers in Neurology

Article Title: Repulsive Guidance Molecule a Inhibits Angiogenesis by Downregulating VEGF and Phosphorylated Focal Adhesion Kinase In Vitro

doi: 10.3389/fneur.2017.00504

Figure Lengend Snippet: Repulsive guidance molecule a (RGMa) inhibited angiogenesis in vitro via neogenin. (A) Human umbilical artery endothelial cells (HUAECs) were transfected with CRISPR/Cas9 neogenin knockout kit and purified with puromycin, then the result of neogenin knockout was validated with western blot. (B,C) HUAECs transfected with SgRNA or neogenin gRNA were treated with vehicle, RGMa, VEGF, or VEGF plus RGMa. The focal adhesion kinase (FAK) (Tyr397) phosphorylation was measured with western blot. (D–G) Migration and (H–K) tube formation of HUAECs transfected with SgRNA or neogenin gRNA were determined by scratch, transwell, and Matrigel tube-formation assays. The relative number of tubes, tube area, and tube length were analyzed with Image J. Scale bar, 100 µm. Data shown are representative of experimental and quantitative results. N ≥ 4 independent experiments. Bars represent mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Recombinant human RGMa, recombinant rat RGMa, recombinant human VEGF, and recombinant rat VEGF were obtained from R&D Systems (MN, USA).

Techniques: In Vitro, Transfection, CRISPR, Knock-Out, Purification, Western Blot, Phospho-proteomics, Migration

Journal: Frontiers in Neurology

Article Title: Repulsive Guidance Molecule a Inhibits Angiogenesis by Downregulating VEGF and Phosphorylated Focal Adhesion Kinase In Vitro

doi: 10.3389/fneur.2017.00504

Figure Lengend Snippet: Unc5b is involved in the effect of repulsive guidance molecule a (RGMa) on phosphorylation of focal adhesion kinase (FAK), migration, and tube formation in human umbilical artery endothelial cells (HUAECs). (A) HUAECs were transfected with CRISPR/Cas9 Unc5b knockout kits and purified with puromycin, then the effect of Unc5b knockout was validated with western blot. (B,C) HUAECs transfected with SgRNA or Unc5b gRNA were treated with control, RGMa, VEGF, or VEGF plus RGMa. FAK (Tyr397) phosphorylation was measured with western blot. (D–G) Migration and (H–K) tube formation of HUAECs transfected with SgRNA or Unc5b gRNA were determined with scratch, transwell, and Matrigel tube-formation assays. The relative number of tubes, tube area, and tube length was analyzed with Image J. Scale bar, 100 µm. Data shown are representative of experimental and quantitative results. N ≥ 4 independent experiments. Bars represent mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Recombinant human RGMa, recombinant rat RGMa, recombinant human VEGF, and recombinant rat VEGF were obtained from R&D Systems (MN, USA).

Techniques: Phospho-proteomics, Migration, Transfection, CRISPR, Knock-Out, Purification, Western Blot, Control

Journal: Frontiers in Neurology

Article Title: Repulsive Guidance Molecule a Inhibits Angiogenesis by Downregulating VEGF and Phosphorylated Focal Adhesion Kinase In Vitro

doi: 10.3389/fneur.2017.00504

Figure Lengend Snippet: Repulsive guidance molecule a (RGMa) inhibited cytoskeleton reassembly, filopodia, and lamellipodia formation in human umbilical artery endothelial cells (HUAECs) via neogenin and Unc5b. Before the immunofluorescence experiment, HUAECs were treated with vehicle, RGMa, VEGF, or VEGF plus RGMa for 40 min. F-actin was stained with phalloidin conjugated with FITC and phosphorylated focal adhesion kinase (p-FAK) connected with primary antibody was labeled with Alexa Fluor 555 donkey anti-rabbit (H + L) secondary antibody. (A) Immunofluorescence showed the cytoskeleton (green) change and p-FAK (red) distribution. (B) Immunofluorescence showing the cytoskeleton change of HUAECs transfected with SgRNA or neogenin gRNA. (C) Immunofluorescence showed the cytoskeleton change of HUAECs transfected with SgRNA or Unc5b gRNA. The filopodia are indicated as sharp spikes (arrowhead), and lamellipodia (arrow) are indicated as flat intensive staining. The merged images are shown in the upper panels, and the amplified indicated areas are shown in the lower panel for different groups. Photographs were obtained with laser scanning confocal microscopy (Nikon, A1 + R, magnification 400×). Results shown are representative images of ≥4 independent experiments.

Article Snippet: Recombinant human RGMa, recombinant rat RGMa, recombinant human VEGF, and recombinant rat VEGF were obtained from R&D Systems (MN, USA).

Techniques: Immunofluorescence, Staining, Labeling, Transfection, Amplification, Confocal Microscopy

Journal: International journal of molecular sciences

Article Title: Finerenone, a Non-Steroidal Mineralocorticoid Receptor Antagonist, Reduces Vascular Injury and Increases Regulatory T-Cells: Studies in Rodents with Diabetic and Neovascular Retinopathy.

doi: 10.3390/ijms24032334

Figure Lengend Snippet: Figure 3. Finerenone reduced VEGF and vascular leakage in (mRen-2)27 after 12 weeks of diabetes. D, diabetic. Veh, vehicle. Per, perindopril. Fin, finerenone. (A) Three-micrometer paraffin sections of retina immunolabeled with VEGF and counterstained with hematoxylin. GCL, ganglion cell layer. IPL, inner plexiform layer. INL, inner nuclear layer. Scale bar = 40 µm. Ganglion cells denoted by single asterisk (*). Glial cells denoted by double asterisks (**). (B) Quantitation of VEGF immunolabeling in the retina. * p < 0.05 to diabetic+vehicle. n = 4 to 5 rats per group. (C) Vitreal levels of albumin (ELISA). * p < 0.05 to non-diabetic and diabetic+vehicle. # p < 0.05 to non-diabetic. n = 7 to 8 rats per group. Values are mean ± SEM.

Article Snippet: Immunohistochemistry for VEGF was performed by incubating three-micrometer paraffin sections with normal donkey serum for 1 h (D9663, Sigma-Aldrich), and then goat anti-rat VEGF (1:500, 564-RV, R&D Systems, Minneapolis, Minnesota, USA) overnight at 4 ◦C.

Techniques: Immunolabeling, Quantitation Assay, Enzyme-linked Immunosorbent Assay

Journal: International journal of molecular sciences

Article Title: Finerenone, a Non-Steroidal Mineralocorticoid Receptor Antagonist, Reduces Vascular Injury and Increases Regulatory T-Cells: Studies in Rodents with Diabetic and Neovascular Retinopathy.

doi: 10.3390/ijms24032334

Figure Lengend Snippet: Figure 5. Finerenone reduced retinal neovascularization, VEGF, and vascular leakage in mice with OIR at postnatal day 18. RA, room air control. Veh, vehicle. Fin, finerenone. (A) Retinal wholemounts labeled with FITC-isolectin to delineate retinal blood vessels in green. Top panels show whole retina. A quadrant of retina (yellow box) is magnified in the lower panel. Scale bar = 500 µm. Asterisks denote vaso-obliteration. Arrows denote neovascularization. (B) Quantitation of retinal neovascularization. * p < 0.05 to OIR+vehicle. n = 5 to 7 mice per group from 2 to 3 liters per group. (C) Retinal VEGF protein levels (ELISA). (D) Retinal VEGF mRNA levels. (E) Retinal vascular leakage (albumin ELISA). ** p < 0.01, **** p < 0.0001 to RA. # p < 0.05, ### p < 0.001 to OIR + vehicle (Kruskal–Wallis test). n = 5 to 8 mice per group from 2 to 3 litters of mice per group. Values are mean ± SEM.

Article Snippet: Immunohistochemistry for VEGF was performed by incubating three-micrometer paraffin sections with normal donkey serum for 1 h (D9663, Sigma-Aldrich), and then goat anti-rat VEGF (1:500, 564-RV, R&D Systems, Minneapolis, Minnesota, USA) overnight at 4 ◦C.

Techniques: Control, Labeling, Quantitation Assay, Enzyme-linked Immunosorbent Assay

Journal: Exploration of BioMat-X

Article Title: Top2b regulates morphological and migratory properties of retinal progenitor cells in vivo and upon transplantable matrix substrates

doi: 10.37349/ebmx.2025.101335

Figure Lengend Snippet: Figure 6. Cultured retinal progenitor cell groups with Top2b overexpression (OE) and knockdown (KD) illustrate altered expression of select chemotactic receptors compared to wildtype cell groups with no Top2b manipulation (WT). Cell groups illustrated significant differences in the expression of selected chemotactic receptors: vascular endothelial growth factor receptor (VEGFR1), fibroblast growth factor receptor (FGFR1), and c-x-c chemokine receptor 4 (CXCR4, receptor for the stromal cell-derived factor 1, SDF-1a, ligand). Statistical significance across the cell groups is denoted by p-value: * < 0.05, ** < 0.01, **** < 0.0001 as via two-way ANOVA tests followed by Dunnett’s post-hoc test

Article Snippet: Approximately 600 μL of each growth factor vascular endothelial growth factor (VEGF; R&D Systems, Minneapolis, MN, USA, 564-RV), fibroblast growth factor-8 (FGF-8; Invitrogen, Carlsbad, CA, USA, PHG0184), and stromal derived factor 1-a (SDF-1a; Sigma Aldrich, St. Louis, MO, USA, SRP3276) was added to the bottom of separate transwell assays.

Techniques: Cell Culture, Over Expression, Knockdown, Expressing, Derivative Assay