Snail is upregulated under extracellular matrix (ECM)-mediated signals.

<p>(A) Western blot analysis showing Snail expression on immobilized ECM. After HUVECs were transfected with siCon or siSnail, the transfectants were reseeded and cultured on PLL (20 μg/mL)-, FN (20 μg/mL)-, or CI (20 μg/mL)-coated culture dishes for 2 h. PLL, poly-L-lysine; FN, fibronectin; CI, collagen type I. (B) Time-course expression pattern of Snail on immobilized ECM. Confluent HUVECs were reseeded and cultured on PLL-, FN- or CI-coated dishes for the indicated time points. Snail expression was evaluated by western blot (upper) and quantitative RT-PCR (lower) analyses. (C) Western blot analysis showing the induction of phosphorylated Akt (p-Akt) and phosphorylated extracellular-regulated kinase 1/2 (p-Erk1/2) in HUVECs that were cultured on FN-coated dishes. (D) Snail expression on immobilized ECM after MK2206 treatment. Confluent HUVECs or human retinal endothelial cells (HRECs) were pre-exposed to 10 μM PP2 (a Src kinase inhibitor) or 1 μg/mL MK2206 (an allosteric Akt inhibitor) for 1 h, followed by reseeding and culture on PLL-, FN-, or CI-coated dishes for 2 h (western blot) or 1 h (quantitative RT-PCR).</p

Snail upregulates VEGF receptor 3 (VEGFR3).

<p>(A) Western blot and RT-PCR analyses showing Snail, early growth response protein-1 (Egr-1), VEGF receptor 3 (VEGFR3), and VEGFR2 expression. HRECs were seeded at a density of 2–2.5×10⁴ cells/cm² on FN- (for western blot and RT-PCR) or PLL (for western blot)-coated dishes and cultured for the indicated time points. (B) Western blot analysis showing the effect of Snail knockdown on VEGFR3. HRECs were reseeded after transfections with siCon or siSnail on FN-coated dishes, and cultured for the indicated time. (C) Quantitative RT-PCR analysis showⁱng the effect of Snail knockdown on VEGFR3 expression. SiSnail-transfected ECs were reseeded and cultured on FN-coated dishes for 8 h. *, p<0.01. (D) Western blot and quantitative RT-PCR analyses showing the effect of Snail overexpression on VEGFR3. HUVECs were transfected with Snail. On the next day, the medium was changed, and the transfected cells were cultured for 8 h (quantitative RT-PCR; right) or 16 h (western blot; left). *, p<0.01.</p

Snail upregulates VEGFR3 transcripts via cooperating with Egr-1.

<p>(A) <i>VEGFR3</i> promoter activity after the exposure of HRECs to immobilized FN. HRECs were transfected with the human <i>VEGFR3</i> promoter_luciferase (hVEGFR3_Luc) reporter (wildR3) and then reseeded at a density of 2–2.5×10⁴ cells/cm² on FN-coated dishes. (B) Schematic illustration of the location of putative Snail and the Egr-1-binding site in the human <i>VEGFR3</i> promoter. WildR3, human <i>VEGFR3</i> promoter_luciferase (hVEGFR3_Luc) reporter; mutR3(Snail), mutation in the putative E-box. Broken line, Egr-1-binding elements; thick line, putative E-box; Luc, luciferase. (C) Western blot analysis showing the effect of Egr-1 knockdown on VEGFR3. HRECs were reseeded after transfections with siCon or siEgr-1 on FN-coated dishes. Arrow, an Egr-1 band; *, a non-specific band. (D) <i>VEGFR3</i> promoter activity after the knockdown of Snail or Egr-1. HRECs were co-transfected with the indicated siRNA and the wildR3 reporter and then reseeded and cultured on FN-coated dishes for 16 h. (E) Mutant <i>VEGFR3</i> promoter activity. HRECs were transfected with the indicated wildR3 and mutR3 (Snail) reporters and then reseeded on FN-coated dishes for 16 h. (F) Immunoprecipitation assay demonstrating the complex association between Snail and Egr-1. HRECs were seeded on FN-coated dishes. After 2 h, the cell lysates were immunoprecipitated (IP) with immunoglobulin G (IgG) or anti-Egr-1 antibody (α-Egr-1). (G) Chromatin immunoprecipitation analysis of the <i>VEGFR3</i> promoter in HUVECs. HUVECs were transfected with flag-Snail (Snail) and immunoprecipitated using anti-Snail antibodies (α-Snail). PCR was performed to detect the <i>VEGFR3</i> promoter region containing the putative E box.</p

Proposed model of capillary branching morphogenesis in postnatal mice.

<p>(A) Outline of Snail stabilization by ECM-mediated signaling. Snail is rapidly degraded by the GSK3β-dependent proteosomal system. On exposure of ECs to ECM, they activate Akt, which can suppress GSK3β-dependent system by phosphorylating GSK3β (pGSK3β). This process stabilizes Snail by releasing it from GSK3β system. Thereby, the formation of Snail-Egr-1 complex promotes VEGFR3 expression by binding to the <i>VEGFR3</i> promoter region to facilitate EC morphogenesis, such as EC sprouting, extension, and branching. pSnail, phosphorylated Snail by GSK3β; pAkt, Akt phosphorylation; E, Egr-1; EC, endothelial cell. (B) Capillary branching morphogenesis is controlled by Snail. In P7–P8 mice, venous ECs in the superficial plexus start to extend capillary branching toward the deep retina in response to tissue needs. The sprouting ECs at the border between the GCL and IPL are exposed to ECM, which subsequently contributes to Snail induction and stabilization, followed by enhanced VEGFR3 expression. Snail/VEGFR3-expressing ECs vertically migrate toward deep retina. At P9–P11 mice, vertically migrating ECs reach in the boundary of INL and turn sideways to form the deep capillary plexus in the OPL region. Snail knockdown attenuates the initiation of EC sprouting, which subsequently impairs the formation of the deep capillary plexus.</p

Endothelial Snail Regulates Capillary Branching Morphogenesis via Vascular Endothelial Growth Factor Receptor 3 Expression

<div><p>Vascular branching morphogenesis is activated and maintained by several signaling pathways. Among them, vascular endothelial growth factor receptor 2 (VEGFR2) signaling is largely presented in arteries, and VEGFR3 signaling is in veins and capillaries. Recent reports have documented that Snail, a well-known epithelial-to-mesenchymal transition protein, is expressed in endothelial cells, where it regulates sprouting angiogenesis and embryonic vascular development. Here, we identified Snail as a regulator of VEGFR3 expression during capillary branching morphogenesis. Snail was dramatically upregulated in sprouting vessels in the developing retinal vasculature, including the leading-edged vessels and vertical sprouting vessels for capillary extension toward the deep retina. Results from <i>in vitro</i> functional studies demonstrate that Snail expression colocalized with VEGFR3 and upregulated <i>VEGFR3</i> mRNA by directly binding to the <i>VEGFR3</i> promoter via cooperating with early growth response protein-1. Snail knockdown in postnatal mice attenuated the formation of the deep capillary plexus, not only by impairing vertical sprouting vessels but also by downregulating VEGFR3 expression. Collectively, these data suggest that the Snail-VEGFR3 axis controls capillary extension, especially in vessels expressing VEGFR2 at low levels.</p></div

Simultaneous Activity Assay of Two Transglutaminase Isozymes, Blood Coagulation Factor XIII and Transglutaminase 2, by Use of Fibrinogen Arrays

We developed an on-chip activity assay system to simultaneously determine the transamidating activities of blood coagulation factor XIII (FXIII) and transglutaminase 2 (TG2) by use of fibrinogen arrays. FXIII and TG2 are transglutaminase family members that are involved in various physiological functions, including vascular pathophysiology, bone development, and cancer progression. However, investigation of their differential functions is limited by the lack of high-throughput and isozyme-specific activity assays. For the on-chip activity assay, we fabricated protein arrays by immobilizing fibrinogen onto the 3-aminopropyltrimethoxysilane surface of well-type arrays, and we determined transamidating activity by probing biotinylated fibrinogen with Cy3-conjugated streptavidin on arrays. We optimized assay conditions, such as buffer pH, concentrations of dithiothreitol and 5-(biotinamido)pentylamine, and incubation time, and we created equations to determine specific FXIII and TG2 activities in samples. We successfully applied this assay system to monitor changes in FXIII and TG2 activities in THP-1 monocytic cells differentiated with phorbol 12-myristate13-acetate and interleukin-4. This activity assay is sensitive and suitable for high-throughput determination of FXIII and TG2 activities and thus has a strong potential for investigating the differential functions of these isozymes in cell signaling and cardiovascular pathophysiology research

Snail is expressed in sprouting vessels.

<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

Vertically sprouting vessels have strong VEGFR3, but weak VEGFR2, expression in the developing retinal vasculature.

<p>(A) Cross-sectional confocal images showing the differential expression pattern of VEGFR2 and VEGFR3 in P11 mice. The immunoreactivity of VEGFR3 was strongly detected in the vertical vessels (IPL and INL; triangles) and deep plexus (OPL, triangles). In contrast, strong immunoreactivity of VEGFR2 was detected in the superficial plexus (GCL, arrows) and neurons (arrow heads). Nuclei were DAPI positive (blue). ONL, outer nuclear layer. Bar, 100 μm. (<b>B</b> and <b>C</b>) Confocal images of VEGFR3 staining in the superficial plexus at P8. Eyeballs from P8 mice were applied to whole flat-mount staining of iB4 and VEGFR3. The region in the box (B) is magnified in <b>C</b> (upper). The region of the vertical vessel was taken below the superficial plexus. (C, lower) The immunoreactivity of VEGFR3 was detected in sprouting vessels from the vein (arrows). Broken lines correspond to the position of vein that appeared in the superficial plexus. A, artery; V, vein. Nuclei were DAPI positive (blue). Bar, 100 μm.</p

Snail knockdown attenuates VEGFR3 expression in the vertical vessel and the deep plexus.

<p>(A) Whole flat-mount images showing the colocalization of Snail and VEGFR3. The immunoreactivity of Snail (green) was observed in the sprouting vessel from the vein in P8 retinal vessels. VEGFR3 immunoreactivity (magenta) was also found in the vein. V, vein. (B) Confocal images of iB4 combined with VEGFR3 staining in shCon or shSnail lentivirus-infected retinas at P8. Mice were consecutively injected intraperitoneally with the shCon or shSnail lentivirus at P6 and P7, as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005324#pgen.1005324.g006" target="_blank">Fig 6A</a>. The shSnail lentivirus was the same virus that was described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005324#pgen.1005324.g001" target="_blank">Fig 1F</a> (shSnail#2). Images of vertical vessels from superficial plexus were taken. Arrows indicate sprouting vessels from veins. Broken line indicates the position of veins in the superficial plexus. Bar, 100 μm. (C) Confocal images of iB4 combined with VEGFR3 staining in the region of the deep capillary plexus in siCon- or siSnail-injected mice at P11. SiRNA injections were performed, as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005324#pgen.1005324.g006" target="_blank">Fig 6A</a>. Cell nuclei were stained with DAPI (blue). The immunoreactivity of iB4 and VEGFR3 was weaker in siSnail-injected mice than in siCon mice. Bar, 100 μm. (D) Quantification of total vessel area and VEGFR3-positive regions in the deep plexus at P11. Over six fields were analyzed. *, p<0.01. (E) Fibrin bead assay. HRECs were transfected with mock and Snail in a combination with VEGFR3 siRNA (siVEGFR3). Representative spheroids are shown for each condition (left). Sprouting numbers per bead, sprouting lengths from each bead, and branch numbers were calculated to quantify endothelial sprouting (right). *, p<0.01.</p

Snail knockdown attenuates retinal vessel sprouting and deep capillary plexus formation.

<p>(A) Illustration of the siRNA or shRNA injection strategy in mice. Mice were consecutively and intraperitoneally injected from P6 to P7 or from P7 to P10 and then sacrificed at P8-P9 (P8/P9) or P11, respectively. (B) Quantitative RT-PCR demonstrating Snail knockdown at P11 in siSnail-injected mice. (C) Confocal images of iB4 staining in the superficial plexus and deep plexus. SiSnail or siCon injection was performed, as described in <b>A</b>. Whole flat-mount retinas were stained with iB4 at P11. Confocal images were taken in the superficial plexus and then taken in the deep plexus below the superficial plexus by moving the z axis of the confocal microscopic field. The formation of the deep plexus was decreased by Snail knockdown. (D) Representative confocal images of iB4 staining at P11 in siCon- and siSnail-injected mice. SiSnail or siCon injection performed, as described in <b>A</b>. Broken lines indicate the position of veins in the superficial plexus. Arrows indicate sprouting vertical vessels from veins in the superficial plexus. Bar, 100 μm. (E) Quantification of vertical vessels and branching points in the deep plexus at P11. *, p<0.05. (F) Confocal images were collected in 1-μm z-stacks in the xz axis at P11 in siCon- and siSnail-injected mice. S.P., the superficial plexus; D.P., the deep plexus.</p