DAL impairs vessel development and regeneration in zebrafish. (A) Macroscopic view of 48 h after fertilization Tg(fli1:EGFP)^y1 zebrafish embryos, comparing control embryos and embryos treated with DAL (5 μM). Treated embryos failed to form vessel branches. (B and C) Regeneration experiments with zebrafish, depicting the vasculature of the caudal fin on 2 and 9 d after amputation in control and treated zebrafish. White lines indicate the amputation plane. (B) Vascular plexus formation, plexus remodeling, and late regenerative angiogenesis. (C) A higher magnification of the reconnected vessels. Nine days after amputation, the regenerating vasculature of each fin ray (three fin rays are shown) consists of a plexus with dense unstructured vessels extending distally from the amputation plane (white arrowheads). Regenerating blood vessels in the wild-type control (fli1:EGFP) form plexuses. Fish treated with DAL have defects in anastomosis and plexus formation. (Scale bars: 200 μm.)

DAL interferes with adhesive properties of endothelial cells in vitro. (A) DAL is involved in actin cytoskeleton assembly regulation. For F-actin visualization, we plated HUVECs and treated them with saline or DAL (10 μM) for 3 h. After fixing and permeabilizing these cultured cells, we subsequently stained them with phalloidin. (B) DAL interferes with adherens junctions in networks of HUVEC cells cultured on matrigel. After 24 h, control HUVEC cells organized into a network of cordlike structures, whereas DAL-treated HUVECs show disjoined networks. Existing cordlike structures, when exposed to 10 μM of DAL for 3 h, were also disrupted. (C) A wound-healing motility assay with HUVECs. We disrupted HUVEC monolayers mechanically with a pipette tip and followed the migration of cells to “heal” the wound using microscopy. DAL (5 μM) slowed healing compared with the control, shown at 24 h after wounding.