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

Park JA, Kim DY, Kim YM, Lee IK, Kwon YG - PLoS Genet. (2015)

Bottom Line: Results from in vitro functional studies demonstrate that Snail expression colocalized with VEGFR3 and upregulated VEGFR3 mRNA by directly binding to the VEGFR3 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, College of Life Science and Biotechnology, Yonsei University, Seoul, Korea.

ABSTRACT
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 in vitro functional studies demonstrate that Snail expression colocalized with VEGFR3 and upregulated VEGFR3 mRNA by directly binding to the VEGFR3 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.

No MeSH data available.


Related in: MedlinePlus

Snail is expressed in sprouting vessels.(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 in vitro 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.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4493050&req=5

pgen.1005324.g001: Snail is expressed in sprouting vessels.(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 in vitro 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.

Mentions: Affymetrix oligonucleotide arrays (GRE accession number GSE12891) were used to compare the mRNA levels of global genes at time points that corresponded to dramatic morphological changes during vascular morphogenesis. Specifically, we looked for genes that were altered during EC network formation, because they may influence endothelial morphological changes in response to cell-cell and cell-ECM interactions (S1 Fig). Snail and Slug expression levels were dramatically increased in those processes. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and western blot analyses confirmed that Snail mRNA and protein levels were dramatically increased at 1 and 2 h when the behavior of ECs was robust (Figs 1A and S1A). At 4 h when vascular network formation was complete, Snail expression disappeared. Although Slug mRNA expression dramatically increased, Slug protein levels could not be detected, thus suggesting that Slug protein is highly unstable during vascular network formation (Fig 1A, middle and right). Furthermore, we found that ectopic expression of Slug in human umbilical vein ECs (HUVECs) dramatically increased Snail, which suggests that Slug could be upstream of Snail (Fig 1B). Similar to our finding, Slug has been reported to be indirectly involved in epithelial branching via Snail upregulation [13]. The differential function between Snail and Slug has been suggested, such that Slug is predominantly effective in cell survival, whereas Snail is involved in invasive and migrating events. Hence, we focused on the role of Snail in the angiogenic process, although Snail and Slug appear to play roles in vascular morphogenesis.


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

Park JA, Kim DY, Kim YM, Lee IK, Kwon YG - PLoS Genet. (2015)

Snail is expressed in sprouting vessels.(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 in vitro 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.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4493050&req=5

pgen.1005324.g001: Snail is expressed in sprouting vessels.(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 in vitro 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.
Mentions: Affymetrix oligonucleotide arrays (GRE accession number GSE12891) were used to compare the mRNA levels of global genes at time points that corresponded to dramatic morphological changes during vascular morphogenesis. Specifically, we looked for genes that were altered during EC network formation, because they may influence endothelial morphological changes in response to cell-cell and cell-ECM interactions (S1 Fig). Snail and Slug expression levels were dramatically increased in those processes. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and western blot analyses confirmed that Snail mRNA and protein levels were dramatically increased at 1 and 2 h when the behavior of ECs was robust (Figs 1A and S1A). At 4 h when vascular network formation was complete, Snail expression disappeared. Although Slug mRNA expression dramatically increased, Slug protein levels could not be detected, thus suggesting that Slug protein is highly unstable during vascular network formation (Fig 1A, middle and right). Furthermore, we found that ectopic expression of Slug in human umbilical vein ECs (HUVECs) dramatically increased Snail, which suggests that Slug could be upstream of Snail (Fig 1B). Similar to our finding, Slug has been reported to be indirectly involved in epithelial branching via Snail upregulation [13]. The differential function between Snail and Slug has been suggested, such that Slug is predominantly effective in cell survival, whereas Snail is involved in invasive and migrating events. Hence, we focused on the role of Snail in the angiogenic process, although Snail and Slug appear to play roles in vascular morphogenesis.

Bottom Line: Results from in vitro functional studies demonstrate that Snail expression colocalized with VEGFR3 and upregulated VEGFR3 mRNA by directly binding to the VEGFR3 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, College of Life Science and Biotechnology, Yonsei University, Seoul, Korea.

ABSTRACT
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 in vitro functional studies demonstrate that Snail expression colocalized with VEGFR3 and upregulated VEGFR3 mRNA by directly binding to the VEGFR3 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.

No MeSH data available.


Related in: MedlinePlus