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Intramembrane binding of VE-cadherin to VEGFR2 and VEGFR3 assembles the endothelial mechanosensory complex.

Coon BG, Baeyens N, Han J, Budatha M, Ross TD, Fang JS, Yun S, Thomas JL, Schwartz MA - J. Cell Biol. (2015)

Bottom Line: We now show that the transmembrane domain of VE-cadherin mediates an essential adapter function by binding directly to the transmembrane domain of VEGFR2, as well as VEGFR3, which we now identify as another component of the junctional mechanosensory complex.Furthermore, VEGFR3 expression is observed in the aortic endothelium, where it contributes to flow responses in vivo.In summary, this study identifies a novel adapter function for VE-cadherin mediated by transmembrane domain association with VEGFRs.

View Article: PubMed Central - HTML - PubMed

Affiliation: Yale Cardiovascular Research Center and Department of Internal Medicine, Cardiovascular Medicine, Yale University School of Medicine, New Haven, CT 06510 Yale Cardiovascular Research Center and Department of Internal Medicine, Cardiovascular Medicine, Yale University School of Medicine, New Haven, CT 06510.

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VEGFRs in shear-induced alignment. HUVECs were transfected with siRNAs as in Fig 4 A before being infected with mCherry (−) or VEGFR-expressing adenoviruses as indicated. Cells were subject to 12 dynes/cm2 laminar shear stress for 16 h. Then, fixed cell alignment was quantified as in Fig 1 B. Values are means ± SEM (error bars), n ≥ 3. *, P < 0.05 relative to siScramble by one-way ANOVA. The broken line indicates random alignment, as in Fig. 1.
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fig6: VEGFRs in shear-induced alignment. HUVECs were transfected with siRNAs as in Fig 4 A before being infected with mCherry (−) or VEGFR-expressing adenoviruses as indicated. Cells were subject to 12 dynes/cm2 laminar shear stress for 16 h. Then, fixed cell alignment was quantified as in Fig 1 B. Values are means ± SEM (error bars), n ≥ 3. *, P < 0.05 relative to siScramble by one-way ANOVA. The broken line indicates random alignment, as in Fig. 1.

Mentions: When alignment after 16 h of flow was assayed, depletion of either VEGFR2 or VEGFR3 substantially inhibited alignment, whereas depletion of both receptors slightly increased the degree of inhibition (Fig. 6). Next, depletion was rescued using adenoviruses containing mCherry (control, −), VEGFR2-GFP, or VEGFR3-GFP to express each paralogue at levels close to endogenous (Fig. S5 A). VEGFR2-GFP rescued not only its own knockdown but also knockdown of VEGFR3; similarly, VEGFR3-GFP rescued both its own and VEGFR2 knockdown (Fig. 6). These results show that while both VEGFR2 and VEGFR3 contribute to flow signaling, they are functionally redundant. Different downstream pathways show different dose requirements, but the effects appear to be essentially additive. In support of this conclusion, we estimated the ratio of endogenous VEGFR2/VEGFR3 in HUVEC to be ∼2:1 by calibrating anti-VEGFR antibodies with VEGFR2-GFP and VEGFR3-GFP constructs, with anti-GFP as a reference antibody (Fig. S5 B).


Intramembrane binding of VE-cadherin to VEGFR2 and VEGFR3 assembles the endothelial mechanosensory complex.

Coon BG, Baeyens N, Han J, Budatha M, Ross TD, Fang JS, Yun S, Thomas JL, Schwartz MA - J. Cell Biol. (2015)

VEGFRs in shear-induced alignment. HUVECs were transfected with siRNAs as in Fig 4 A before being infected with mCherry (−) or VEGFR-expressing adenoviruses as indicated. Cells were subject to 12 dynes/cm2 laminar shear stress for 16 h. Then, fixed cell alignment was quantified as in Fig 1 B. Values are means ± SEM (error bars), n ≥ 3. *, P < 0.05 relative to siScramble by one-way ANOVA. The broken line indicates random alignment, as in Fig. 1.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4384728&req=5

fig6: VEGFRs in shear-induced alignment. HUVECs were transfected with siRNAs as in Fig 4 A before being infected with mCherry (−) or VEGFR-expressing adenoviruses as indicated. Cells were subject to 12 dynes/cm2 laminar shear stress for 16 h. Then, fixed cell alignment was quantified as in Fig 1 B. Values are means ± SEM (error bars), n ≥ 3. *, P < 0.05 relative to siScramble by one-way ANOVA. The broken line indicates random alignment, as in Fig. 1.
Mentions: When alignment after 16 h of flow was assayed, depletion of either VEGFR2 or VEGFR3 substantially inhibited alignment, whereas depletion of both receptors slightly increased the degree of inhibition (Fig. 6). Next, depletion was rescued using adenoviruses containing mCherry (control, −), VEGFR2-GFP, or VEGFR3-GFP to express each paralogue at levels close to endogenous (Fig. S5 A). VEGFR2-GFP rescued not only its own knockdown but also knockdown of VEGFR3; similarly, VEGFR3-GFP rescued both its own and VEGFR2 knockdown (Fig. 6). These results show that while both VEGFR2 and VEGFR3 contribute to flow signaling, they are functionally redundant. Different downstream pathways show different dose requirements, but the effects appear to be essentially additive. In support of this conclusion, we estimated the ratio of endogenous VEGFR2/VEGFR3 in HUVEC to be ∼2:1 by calibrating anti-VEGFR antibodies with VEGFR2-GFP and VEGFR3-GFP constructs, with anti-GFP as a reference antibody (Fig. S5 B).

Bottom Line: We now show that the transmembrane domain of VE-cadherin mediates an essential adapter function by binding directly to the transmembrane domain of VEGFR2, as well as VEGFR3, which we now identify as another component of the junctional mechanosensory complex.Furthermore, VEGFR3 expression is observed in the aortic endothelium, where it contributes to flow responses in vivo.In summary, this study identifies a novel adapter function for VE-cadherin mediated by transmembrane domain association with VEGFRs.

View Article: PubMed Central - HTML - PubMed

Affiliation: Yale Cardiovascular Research Center and Department of Internal Medicine, Cardiovascular Medicine, Yale University School of Medicine, New Haven, CT 06510 Yale Cardiovascular Research Center and Department of Internal Medicine, Cardiovascular Medicine, Yale University School of Medicine, New Haven, CT 06510.

Show MeSH
Related in: MedlinePlus