Snail is expressed in sprouting vessels.

Abstract

<p>(A) Quantitative reverse transcription-polymerase chain reaction (RT-PCR) (left and middle) and western blot (right) analyses showing the expression pattern of Snail and Slug during <i>in vitro</i> vascular network formation. Human umbilical vein endothelial cells (HUVECs) were placed on Matrigel and analyzed at the indicated time points. *, p<0.001. (B) Western blot analysis showing Slug-mediated Snail induction. Slug was transfected with the indicated doses in HUVECs. On the next day, the cells were lysed, and western blot analysis was performed. (C) Illustration of the developing retinal vessel from the superficial plexus to the deep plexus in mice at postnatal day 11 (P11). The superficial plexus is represented by vessels around the ganglion cell layer (GCL), the vertical vessel includes vessels around the inner plexiform layer (IPL) and inner nuclear layer (INL), and the deep plexus is represented by vessels around the outer plexiform layer (OPL). (D) Confocal images showing Snail immunoreactivity. Whole flat-mount staining analysis was performed in eyeballs at P8. The immunoreactivity of Snail (green) was observed in sprouting vessels from the vein. A, artery; V, vein; iB4, isolectin B4. Bar, 100 μm. (E) Cross-sectional confocal images at P11 showing Snail expression in the descending vessels. Sections were stained with anti-Snail (green) and anti-CD31 (red) antibodies. The immunoreactivity of Snail was detected in the superficial branching region (GCL and IPL; arrows) and the vertical vessels (INL; triangles). Bar, 100 μm. (F) Representative images of Matrigel plugs at 6 days after the subcutaneous injection of Matrigel plugs containing the small hairpin (sh)Lenti Snail virus and vascular endothelial growth factor A (VEGFA; 200 ng/mL) into C57BL/6 mice (n = 6 per group). Two types of shLenti Snail virus (shSnail#1 and shSnail#2) were used. (G) Immunohistochemical analysis showing infiltrating mouse CD31⁺ ECs (red). The Matrigel plug containing the shLenti Snail virus (shSnail#2) recruited mouse ECs but failed to initiate vascular network formation. (H) Quantification of vessel ingrowth by measuring CD31⁺ length (right). *, p<0.01. (I) Snail immunofluorescence in a fibrin gel bead after one day of culture. The cells were stained with anti-Snail antibodies (green). Nuclei were DAPI-positive (blue). (J) Immunofluorescence images of the mixed culture of control siCon and siSnail-GFP- transfected HUVECs. SiSnail was transfected in GFP-overexpressing HUVECs, and siCon was transfected in HUVECs before mixed culture (1:1) on fibrin beads. Most of the siSnail-GFP-transfected cells remained on the beads, whereas siCon-transfected cells sprouted to the fibrin gel. siSnail, small-interfering RNA targeting Snail. (K) Fibrin bead assay showing representative images by siCon- and siSnail-transfected HUVECs (left). Sprouting numbers per bead or sprouting lengths from one bead were calculated to quantify endothelial sprouting (right). *, p<0.01.</p