Endosialin modulates VEGF-A signaling and promotes endothelial cell apoptosis. (A) HUVECs cultured on ES-Fc- or hFc-coated plates were stimulated with VEGF-A, lysed, and subject to immunoblotting using the indicated Abs. Antitubulin mAb was used as a loading control. Molecular size markers are in kilodaltons. High- and low-exposure blots are shown. Quantification of the band intensities are from 3 independent experiments ± SEM. Data are normalized to the loading control and are shown relative to the hFc control at each time point. (B) HUVECs (4 × 10³) were cultured on ES-Fc- or hFc-coated 96-well plates for 24 hours. Media were replaced with serum-free media supplemented with 20 ng/mL of VEGF-A and cells were cultured for a further 24 hours. Cell viability was measured using the CellTiter-Glo assay. Data shown are from 1 of 3 representative experiments with n = 3 samples ± SEM. (C) HUVECs or 10T1/2 cells incubated for 3 hours with VEGF-A in the presence of ES-Fc or hFc were stained with FITC–annexin V and/or propidium iodide (PI) and subjected to FACS analysis. Representative HUVEC FACS profiles are shown. Graph shows annexin V–positive/PI-positive, and annexin V–positive/PI-negative cells in the presence of ES-Fc from 2 independent experiments ± SD relative to the hFc control. (D) Phase-contrast images of HUVECs cultured in medium containing VEGF-A in the presence of hFc, ES-Fc, or CD44-Fc for 0, 8, and 20 hours. Scale bar indicates 100 μm. (E) Retinas from Endosialin^+/+, Endosialin^+/−, and Endosialin^−/− C57BL/6 mice were stained for collagen IV (red), activated caspase-3 (white), and FITC–isolectin B4 (green). Images show activated caspase-3–positive endothelial cells (arrowheads) associated with collagen IV empty sleeves in P4 retinas. Scale bar indicates 20 μm. Graph shows relative number of apoptotic (activated caspase-3–positive) vessels ± SEM. Endosialin^+/+: P4, n = 7; P5, n = 2; Endosialin^+/−: P4, n = 6; P5, n = 5; Endosialin^−/−: P4, n = 11; P5, n = 4.