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Vascular endothelial cells that express dystroglycan are involved in angiogenesis

¹ Center for Cardiovascular Research, University of Rochester School of Medicine and Dentistry, Rochester NY 14642-8679, USA
² Department of Neurobiology, Tottori University Faculty of Medicine, Yonago 683, Japan
³ Department of Pathology, Tottori University Faculty of Medicine, Yonago 683, Japan
⁴ National Cardiovascular Center Research Institute, Osaka 565, Japan

View larger version (91K): [in a new window]   Fig. 4. Proliferation of FL-DG- or ßDG-expressing BAE. (A) Photomicrographs of confluent monolayers of cells transfected with vector alone (vector), a full-length DG construct (FL-DG) or a construct of the cytoplasmic tail of DG (ßDG). Cells were photographed after 7 days in culture. (B) Quantification of the cell surface area. The cell surface area was measured from images similar to the ones shown in (A) using an NIH image software. Each bar represents the mean ±s.e.m. of the values from more than 100 cells obtained in three independent experiments. (C) Proliferation curves. Transfected cells were seeded in 24-well plates at a low density and allowed to proliferate in DMEM/10% FCS. The mean numbers of cells of triplicate determinations obtained in a single experiment are shown. Similar results were obtained on two other occasions.

View larger version (54K): [in a new window]   Fig. 1. Expression of FL-DG and ßDG in BAE. (A) Cells were transfected with pREP/F, pREP/C or vector alone and double-stained after geneticin selection with anti-ßDG and Texas-Red—phalloidin. Bound antibodies were visualized by a Cy2-conjugated secondary antibody. Serial optical sections (1 µm intervals) were obtained with a confocal microscope and through-focused images were reconstituted for the anti-ßDG images. At the laser intensity and the window level applied to reveal the distribution of expressed ßDG (FL-DG) and ßDG (ßDG), the signals from endogenous ßDG were extremely low. Subcellular distribution of endogenous ßDG (vector) in BAE has been described in detail (Shimizu et al., 1999). (B,C) Enrichment of FL-DG- or ßDG-expressing BAE. Cells were transfected and selected for resistance against geneticin and then processed for anti-ßDG flow cytometry (B) or western blotting (C). Note that each of the three types of transfected cells forms a single peak, indicating that it is a homogenious cell population (B). Arrowheads in (C) indicate intact ßDG (43 kDa) and ßDG (17 kDa). Molecular weights (kDa) are given on the left. Shown are the results obtained in a single experiment. Similar findings were obtained in two other independent experiments.

View larger version (31K): [in a new window]   Fig. 2. Adhesion of FL-DG- or ßDG-expressing BAE to dishes coated with laminin-1 or fibronectin. Cells were transfected and selected for resistance against geneticin. The adhesion assay was performed as described in the Materials and Methods in the presence or absence of 2 mg/ml heparin. During the early phase of cell attachment (2 hours), the extent of cell spreading on the substrate was similar in all cases. Each bar represents the means±s.e.m. of three determinations, each done in triplicate. The values are expressed relative to the estimated number of the attached vector-transfected cells in the absence of heparin (100%). * p<0.01; significantly different from the values of vector-transfected cells.

View larger version (101K): [in a new window]   Fig. 3. Migration of FL-DG- or ßDG-expressing BAE on laminin-1. Cells transfected with a cDNA construct for vector alone (a), FL-DG (b) or ßDG (c) and genecitin-selected were subjected to the wound-healing assay as described in the Materials and Methods. The initial width of the wound was the distance between the top and the bottom margins of each micrograph. Photographs of cells that migrated into the wound area were taken after 24 hours. Bar, 100 µm.

View larger version (48K): [in a new window]   Fig. 5. Tube formation by BAE expressing FL-DG or ßDG. (A) The same number of cells transfected with vector alone (vectgor) or overexpressing DG (FL-DG) or truncated ßDG (ßDG) were seeded on Matrigel. The phase contrast images of the culture bed at the indicated times are shown. (B) Levels of endogenous ßDG expression in vector-transfected cells. Membrane fractions were prepared from cells cultured on Matrigel for the times indicated. They were analyzed by anti-ßDG western blotting. 10 µg of protein was loaded on each lane. Anti-actin immunoblot is used as loading control. The arrowhead indicates the 43kDa ßDG band. Both in (A) and (B), the results obtained in a single experiment are shown. Similar findings were obtained on two other occasions.

View larger version (19K): [in a new window]   Fig. 6. Changes in expression levels of ßDG in synchronized cultures of BAE as the cells progress through a cell cycle. Cells were arrested at G0/G1 by serum-deprivation and then stimulated with 5% FCS to allow cell cycle progression for up to 24 hours. (A) Cell cycle analysis by flow cytometry. (B) Anti-ßDG western blotting of membrane preparations. Anti-actin immunoblot is used to as loading control. Both in (A) and (B), the results obtained in a single experiment are shown, but similar findings were obtained in two other independent experiments.

View larger version (130K): [in a new window]   Fig. 7. Anti-ßDG immunohistochemistry of surgical human specimens. Bound antibodies were visualized with HRP-conjugated secondary antibody and DAB (brown), and sections were also counterstained with hematoxylin. (A-F) Kidney cancer. Shown are the photomicrographs of cancer tissues (B,D,F) and of normal tissue (A,C,E) that surrounds the tumor. The venules boxed in (A) and (B) are enlarged in (C) and (D), respectively. Arrows point to endothelial cells. The slide from which (E) and (F) were taken was treated with DAB for a longer period of time (see non-specific background staining of red blood cells), but VEC of the vessels found in the normal tissue were not labeled (E). (G,H) Colon cancer. Photomicrographs of arteriole s found in the cancer (H) and the normal tissue (G) that surrounds the tumor are shown.