CD151 up-regulates endothelial cell-matrix adhesiveness. (A) Cell-matrix adhesion assay. HMEC transductants were seeded on ECM substrate-coated wells in triplicates and allowed to settle at 37°C for 35 minutes. Nonadherent cells were then removed by gentle washing. The adhered cells were counted. *P < .01. (B) Traction force microscopy. Cells were plated on FN-, LN 111-, or LN 332-conjugated fluorescent bead-embedded polyacrylamide gels. Traction forces exerted by the cells were measured as described in “Traction force microscopy.” Left: Phase-contrast images and traction field of cells on FN-conjugated polyarylamide gels. Bar represents 10 μm. Right: Maximum and average perimeter traction forces of the HMEC transductants on FN, LN 111, or LN 332. The magnitudes of maximum and average perimeter traction forces were compared with a nonparametric Mann-Whitney test. **P < .05. (C) CD151 silencing attenuates the formation and maturation of focal adhesions. HMEC transductants, after 2-day culture, were fixed, permeabilized, and incubated with vinculin mAb, followed by AlexaFluor-488–conjugated secondary Ab and phalloidin–AlexaFluor-594 staining. Staining was imaged with a Zeiss LSM510 confocal fluorescence microscope under a 100×/1.4 NA oil objective. Bar represents 10 μm. (D) TIRF microscopy. HMEC transductants were fixed without permeabilization, probed with α3 integrin mAb and AlexaFluro-488–conjugated secondary Ab, and visualized by TIRF microscopy. Bar represents 15 μm. The size and numbers of fluorescent particles of α3 integrin and CD9 from the individual transductants (n = 20) were analyzed and compared. (E) Silencing of CD151 expression elevates detergent solubility of β1 integrin. HMEC transductants were lysed with 0.05% Triton X-100 in HEPES buffer. After ultracentrifugation, supernatants were used as soluble fractions. Pellets were solubilized in 1× Laemmli sample buffer and used as the insoluble fractions. Integrin β1 in both fractions as well as whole cell lysates were subjected to SDS-PAGE and then detected by immunoblotting using TS2/16 mAb. Tubulin from whole cell lysates was used as loading control. (F) TS2/16 partially rescues the defects in maintenance of capillary structures in CD151-silenced ECs. HMEC transductants were incubated with 1 μg/mL β1 integrin-activating mAb TS2/16 on ice for 1 hour and were plated on Matrigel in the presence of TS2/16. The capillary-like structures were imaged and quantified. An isotype-matched antihuman CD71 mAb served as the negative control. Bar represents 250 μm. (G) Active β1 integrins were unaltered on CD151 silencing. The levels of activated β1 integrins on HMECs were measured by flow cytometry using mAb AG89.30 (H) The dissociation of ECs from the basement membrane (BM) and splitting of BM in the absence of CD151. Twelve-week-old male CD151 KO (n = 4) and littermate WT (n = 4) mice were perfused and fixed with 2.5% glutaraldehyde. The aortas were isolated, sectioned transversely, and processed for transmission electron microscopy. The red asterisks indicate the space where ECs were detached from BM. Bar represents 250 nm.
