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 upregulates VEGF receptor 3 (VEGFR3).(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.
© Copyright Policy
Related In: Results  -  Collection

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

pgen.1005324.g003: Snail upregulates VEGF receptor 3 (VEGFR3).(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.

Mentions: To determine whether ECM could induce the expression of VEGFRs in ECs, human retinal endothelial cells (HRECs) and HUVECs were exposed to fibronectin. Fibronectin, but not PLL, dramatically increased VEGFR3 mRNA and protein expression (Figs 3A and S4A). Interestingly, Snail knockdown with siRNA reversed ECM-mediated VEGFR3 upregulation at the protein and mRNA levels but showed no effect on VEGFR2 and NRP (Fig 3B and 3C). The increase in VEGFR3 was confirmed by the ectopic expression of Snail (Fig 3D).


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 upregulates VEGF receptor 3 (VEGFR3).(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.
© Copyright Policy
Related In: Results  -  Collection

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

pgen.1005324.g003: Snail upregulates VEGF receptor 3 (VEGFR3).(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.
Mentions: To determine whether ECM could induce the expression of VEGFRs in ECs, human retinal endothelial cells (HRECs) and HUVECs were exposed to fibronectin. Fibronectin, but not PLL, dramatically increased VEGFR3 mRNA and protein expression (Figs 3A and S4A). Interestingly, Snail knockdown with siRNA reversed ECM-mediated VEGFR3 upregulation at the protein and mRNA levels but showed no effect on VEGFR2 and NRP (Fig 3B and 3C). The increase in VEGFR3 was confirmed by the ectopic expression of Snail (Fig 3D).

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