Figure 3.
Endothelial cobblestone morphology and expression of endothelial cell adhesion proteins VE-cadherin and PECAM-1 at cell–cell junctions of ECFCs. (A) The typical endothelial cobblestone morphology of ECFCs isolated from a healthy individual and the IP. Scale bars, 100 µm. (B) VWF (green) and adherence junction protein PECAM-1 (red) are visualized with coimmunostaining of ECFCs derived from a healthy individual (upper) and patient (IP-ECFCs) (lower). Scale bars, 50 µm. (C) The intracellular level of PECAM-1 determined by in-cell ELISA assay (Abcam, UK). Cells of 3 healthy individuals and IP were seeded into collagen precoated 96-well microplate at a density of 2 × 104 cells/100 µL per well. Confluent ECFCs grown into 96-well plates were fixed, permeabilized, and stained with primary (CD31 antibody against PECAM-1) and horseradish peroxidase (HRP)-conjugated secondary antibodies. HRP-conjugated secondary antibodies were goat anti-mouse (Thermo Scientific, UK) and anti-rabbit (Abcam, UK) IgG. After the addition of the HRP-development solution, optical density at 650 (in a kinetic mode, within 60 minutes) was measured using a microplate reader (Synergy 2, BioTek). Last, The HRP signal of antibody-specific complexes was normalized to the Janus Green staining intensity to account for differences in cell density. Measurements were repeated for 3 independent experiments in triplicate (N = 9). Values are means ± SEM. ***P < .001 (unpaired Student t test). (D) ECFCs isolated from a healthy control (left) and the IP (right) were costained with antibodies recognizing VE-cadherin (red) and nucleus (DAPI staining, blue). The IP-ECFCs show defect in the expression of VE-cadherin at endothelial cell–endothelial cell junctions, associated with cytoplasmic red staining, indicating VE-cadherin internalization. Scale bars, 50 µm. (E) The response of the ECFCs to the thrombin stimulation by demonstrating gap formation between cells in the ECFCs monolayer. After thrombin treatment (10 nm, 5 minutes), cells were immunostained with an antibody targeted against VE-cadherin at endothelial cell junctions, and white-gray imaging was done by using inverted fluorescence microscopy. Scale bars, 100 µm. Boxes signify close-up views of VE-cadherin at cell-cell junctions.

Endothelial cobblestone morphology and expression of endothelial cell adhesion proteins VE-cadherin and PECAM-1 at cell–cell junctions of ECFCs. (A) The typical endothelial cobblestone morphology of ECFCs isolated from a healthy individual and the IP. Scale bars, 100 µm. (B) VWF (green) and adherence junction protein PECAM-1 (red) are visualized with coimmunostaining of ECFCs derived from a healthy individual (upper) and patient (IP-ECFCs) (lower). Scale bars, 50 µm. (C) The intracellular level of PECAM-1 determined by in-cell ELISA assay (Abcam, UK). Cells of 3 healthy individuals and IP were seeded into collagen precoated 96-well microplate at a density of 2 × 104 cells/100 µL per well. Confluent ECFCs grown into 96-well plates were fixed, permeabilized, and stained with primary (CD31 antibody against PECAM-1) and horseradish peroxidase (HRP)-conjugated secondary antibodies. HRP-conjugated secondary antibodies were goat anti-mouse (Thermo Scientific, UK) and anti-rabbit (Abcam, UK) IgG. After the addition of the HRP-development solution, optical density at 650 (in a kinetic mode, within 60 minutes) was measured using a microplate reader (Synergy 2, BioTek). Last, The HRP signal of antibody-specific complexes was normalized to the Janus Green staining intensity to account for differences in cell density. Measurements were repeated for 3 independent experiments in triplicate (N = 9). Values are means ± SEM. ***P < .001 (unpaired Student t test). (D) ECFCs isolated from a healthy control (left) and the IP (right) were costained with antibodies recognizing VE-cadherin (red) and nucleus (DAPI staining, blue). The IP-ECFCs show defect in the expression of VE-cadherin at endothelial cell–endothelial cell junctions, associated with cytoplasmic red staining, indicating VE-cadherin internalization. Scale bars, 50 µm. (E) The response of the ECFCs to the thrombin stimulation by demonstrating gap formation between cells in the ECFCs monolayer. After thrombin treatment (10 nm, 5 minutes), cells were immunostained with an antibody targeted against VE-cadherin at endothelial cell junctions, and white-gray imaging was done by using inverted fluorescence microscopy. Scale bars, 100 µm. Boxes signify close-up views of VE-cadherin at cell-cell junctions.

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