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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 knockdown attenuates VEGFR3 expression in the vertical vessel and the deep plexus.(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 Fig 6A. The shSnail lentivirus was the same virus that was described in Fig 1F (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 Fig 6A. 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.
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pgen.1005324.g007: Snail knockdown attenuates VEGFR3 expression in the vertical vessel and the deep plexus.(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 Fig 6A. The shSnail lentivirus was the same virus that was described in Fig 1F (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 Fig 6A. 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.

Mentions: Whole flat-mount staining was performed to assess whether Snail colocalized with VEGFR3 in sprouting vessels (Fig 7A). Expression of VEGFR3 and Snail was high in veins of the GCL at P8, demonstrating that their colocalization could be related to venous sprouting and extension. We thus investigated whether Snail knockdown-induced sprouting defects could be related to VEGFR3 expression in vivo. Knockdown of Snail by shSnail injection at consistent intervals reduced both VEGFR3 immunoreactivity and sprouting in the vein at P8 (Figs 6A and 7B). Results from confocal z-axis analysis demonstrate that Snail knockdown reduced the intensity of iB4 immunoreactivity and VEGFR3 expression in retinas at P11 (Figs 7C, 7D and S8).


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 knockdown attenuates VEGFR3 expression in the vertical vessel and the deep plexus.(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 Fig 6A. The shSnail lentivirus was the same virus that was described in Fig 1F (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 Fig 6A. 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.
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pgen.1005324.g007: Snail knockdown attenuates VEGFR3 expression in the vertical vessel and the deep plexus.(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 Fig 6A. The shSnail lentivirus was the same virus that was described in Fig 1F (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 Fig 6A. 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.
Mentions: Whole flat-mount staining was performed to assess whether Snail colocalized with VEGFR3 in sprouting vessels (Fig 7A). Expression of VEGFR3 and Snail was high in veins of the GCL at P8, demonstrating that their colocalization could be related to venous sprouting and extension. We thus investigated whether Snail knockdown-induced sprouting defects could be related to VEGFR3 expression in vivo. Knockdown of Snail by shSnail injection at consistent intervals reduced both VEGFR3 immunoreactivity and sprouting in the vein at P8 (Figs 6A and 7B). Results from confocal z-axis analysis demonstrate that Snail knockdown reduced the intensity of iB4 immunoreactivity and VEGFR3 expression in retinas at P11 (Figs 7C, 7D and S8).

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