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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 retinal vessel sprouting and deep capillary plexus formation.(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 A. 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 A. 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.
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pgen.1005324.g006: Snail knockdown attenuates retinal vessel sprouting and deep capillary plexus formation.(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 A. 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 A. 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.

Mentions: To examine the role of Snail in the formation of vertical branching and deep capillary plexus, stable Snail siRNA (siSnail) was daily injected into mice from P7 to P10 or from P6 to P8 intraperitoneally (Fig 6A). The efficacy of the siSnail was validated by quantitative RT-PCR at P11 and whole flat-mount analyses at P9 (Figs 6B and S7A). Moreover knockdown of Snail significantly downregulated VEGFR3 expression in whole retinal lysates (Fig 6B). Whole flat-mount analysis showed that the deep plexus was formed from the optic stalk to the retinal margin at P11 (Fig 6C). Snail knockdown impaired the formation of the deep plexus (Figs 6C and S7A, OPL). The distance of the vasculature from the optic stalk to the margin was decreased in siSnail mice. Furthermore, the vertical vessels from the superficial plexus were decreased (Fig 6D, IPL; S7A Fig, GCL). The numbers of vertical vessels that sprouted from the vein were reduced in siSnail retinas and vessel branch points in the deep plexus were also reduced (Figs 6D, 6E, and S7A, IPL and OPL). Confocal z-stack analysis showed the attenuation of vertical vessels in siSnail retinas, compared to siCon retinas (Fig 6F). To avoid the off-target effects of stable siSnail, we utilized the shSnail system, as described in Fig 1F–1H. After intraperitoneal treatments with shShail, whole flat-mount studies showed the reduction in vertical sprouting from the superficial plexus (Figs 6A and S7B). These data demonstrate that Snail played a crucial role in venous vertical sprouting and in the formation of the deep capillary plexus.


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 retinal vessel sprouting and deep capillary plexus formation.(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 A. 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 A. 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.
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pgen.1005324.g006: Snail knockdown attenuates retinal vessel sprouting and deep capillary plexus formation.(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 A. 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 A. 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.
Mentions: To examine the role of Snail in the formation of vertical branching and deep capillary plexus, stable Snail siRNA (siSnail) was daily injected into mice from P7 to P10 or from P6 to P8 intraperitoneally (Fig 6A). The efficacy of the siSnail was validated by quantitative RT-PCR at P11 and whole flat-mount analyses at P9 (Figs 6B and S7A). Moreover knockdown of Snail significantly downregulated VEGFR3 expression in whole retinal lysates (Fig 6B). Whole flat-mount analysis showed that the deep plexus was formed from the optic stalk to the retinal margin at P11 (Fig 6C). Snail knockdown impaired the formation of the deep plexus (Figs 6C and S7A, OPL). The distance of the vasculature from the optic stalk to the margin was decreased in siSnail mice. Furthermore, the vertical vessels from the superficial plexus were decreased (Fig 6D, IPL; S7A Fig, GCL). The numbers of vertical vessels that sprouted from the vein were reduced in siSnail retinas and vessel branch points in the deep plexus were also reduced (Figs 6D, 6E, and S7A, IPL and OPL). Confocal z-stack analysis showed the attenuation of vertical vessels in siSnail retinas, compared to siCon retinas (Fig 6F). To avoid the off-target effects of stable siSnail, we utilized the shSnail system, as described in Fig 1F–1H. After intraperitoneal treatments with shShail, whole flat-mount studies showed the reduction in vertical sprouting from the superficial plexus (Figs 6A and S7B). These data demonstrate that Snail played a crucial role in venous vertical sprouting and in the formation of the deep capillary plexus.

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