Figure 4.

Expression of HAS2 in HUVEC cords and tubes. (A–C) Stages of tube formation in whole-mount, multiaxial vasculogenic cultures, as shown by epifluorescence microscopy in (A) and (B) (red, phalloidin stain for f-actin; blue, DAPI nuclear stain) and bright-field microscopy in (C) (crystal violet stain). At 24 hr (A), cells acquire spindle shapes with long, narrow pseudopodia (e.g., arrows). By 48 hr (B), the cells form a network of multicellular cords (arrows indicate three cords). Appearance of fully developed tubes (one is indicated in C), each with a patent lumen (Lu) and thin walls (arrows), occurs at 3–7 days. Areas of cytoplasm containing nuclei (asterisks) bulge into the lumen. In a Western blot of tubulogenic HUVEC extract (D), anti-HAS2 antibody (green stain) recognizes 53 and 64 kDa bands (asterisks). β-actin (red stain, arrowhead) marks 42 kDa. Ponceau S stain reveals many non-immunoreactive protein bands. (E–G) Whole-mounts of HUVEC cords and tubes in three-dimensional collagen gels evaluated by IF/confocal microscopy for HAS2 (green stain). (E) Cord of a 48-hr culture showing three cells (arrows) at a branch point. (F, G) Cross-sections of large-diameter (F) and small-diameter (G) tubes with patent lumens in a 4-day culture. In (F), arrows indicate the cells comprising the tube wall. In (E)–(G), f-actin is stained with phalloidin (red) and nuclei are stained with TO-PRO-3 (blue). Scale bar sizes: A, B = 100 µm; C = 50 µm; E, F = 10 µm; G = 5 µm. Abbreviations: HUVEC, human umbilical vein endothelial cell; DAPI, 4, 6-diamidino-2-phenylindole; IF, immunofluorescence; DF, dye front; MW, molecular weight.