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 upregulated under extracellular matrix (ECM)-mediated signals.(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).
© Copyright Policy
Related In: Results  -  Collection

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

pgen.1005324.g002: Snail is upregulated under extracellular matrix (ECM)-mediated signals.(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).

Mentions: The developing retinal vasculature into the deep retinal layer is influenced by ECM-mediated integrin signals and retinal neuron-induced hypoxic signals [3,21]. On the basis of the finding that Snail immunoreactivity was detected in the invading vessels into surrounding matrix microenvironment, we investigated the mechanism by which ECM could regulate Snail expression. Fibronectin and collagen type I are major ECM components that are involved in angiogenesis [22]. Exposure of HUVECs to these ECM components dramatically induced Snail protein and mRNA expression (Fig 2A and 2B). In comparison, exposure of HUVECs to poly-L-lysine (PLL), a non-specific adhesion facilitator, only slightly induced Snail protein and mRNA expression. In normal, cultured ECs, Snail protein is unstable and can only be detected in the presence of proteosome inhibitors [10]. Several studies examining the stability of Snail protein have shown that Snail is rapidly degraded via the glycogen synthase kinase (GSK) 3β-dependent proteosomal system in epithelial cells. Activated Akt can phosphorylate GSK3β, and this process stabilizes Snail by releasing it from the GSK3β system [23]. We found that exposure of HUVECs to the ECM induced Akt phosphorylation (Fig 2C). Thus we examined whether the maintenance of Snail protein on ECM component was due to Akt activity. Pretreatment with MK2206 (an allosteric Akt inhibitor) attenuated fibronectin- and collagen type I-mediated Snail induction in protein level (Fig 2D, left and middle). In contrast, mRNA level of Snail showed slight decrease (Fig 2D, right). Although further experiments are required to better understand the transcriptional regulation of Snail by ECM signaling, these results suggest that ECM-induced Snail protein in ECs was stabilized by Akt signals, which prevented Snail from GSK3β-proteosomal degradation.


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 upregulated under extracellular matrix (ECM)-mediated signals.(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).
© Copyright Policy
Related In: Results  -  Collection

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

pgen.1005324.g002: Snail is upregulated under extracellular matrix (ECM)-mediated signals.(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).
Mentions: The developing retinal vasculature into the deep retinal layer is influenced by ECM-mediated integrin signals and retinal neuron-induced hypoxic signals [3,21]. On the basis of the finding that Snail immunoreactivity was detected in the invading vessels into surrounding matrix microenvironment, we investigated the mechanism by which ECM could regulate Snail expression. Fibronectin and collagen type I are major ECM components that are involved in angiogenesis [22]. Exposure of HUVECs to these ECM components dramatically induced Snail protein and mRNA expression (Fig 2A and 2B). In comparison, exposure of HUVECs to poly-L-lysine (PLL), a non-specific adhesion facilitator, only slightly induced Snail protein and mRNA expression. In normal, cultured ECs, Snail protein is unstable and can only be detected in the presence of proteosome inhibitors [10]. Several studies examining the stability of Snail protein have shown that Snail is rapidly degraded via the glycogen synthase kinase (GSK) 3β-dependent proteosomal system in epithelial cells. Activated Akt can phosphorylate GSK3β, and this process stabilizes Snail by releasing it from the GSK3β system [23]. We found that exposure of HUVECs to the ECM induced Akt phosphorylation (Fig 2C). Thus we examined whether the maintenance of Snail protein on ECM component was due to Akt activity. Pretreatment with MK2206 (an allosteric Akt inhibitor) attenuated fibronectin- and collagen type I-mediated Snail induction in protein level (Fig 2D, left and middle). In contrast, mRNA level of Snail showed slight decrease (Fig 2D, right). Although further experiments are required to better understand the transcriptional regulation of Snail by ECM signaling, these results suggest that ECM-induced Snail protein in ECs was stabilized by Akt signals, which prevented Snail from GSK3β-proteosomal degradation.

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