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Updated: Mar 28, 2026
In Vitro Three-Dimensional Sprouting Assay of Angiogenesis Using Mouse Embryonic Stem Cells for Vascular Disease Modeling and Drug Testing
Published on: May 11, 2021
Targeting NCK-Mediated Endothelial Cell Front-Rear Polarity Inhibits Neovascularization
Alexandre Dubrac1, Gael Genet1, Roxana Ola1
1From Cardiovascular Research Center, Yale University School of Medicine, New Haven, CT (A.D., G.G., R.O., F.Z., J.H., J.Z., J.-L.T., A.E.); INSERM U1050, Collège de France, Center for Interdisciplinary Research in Biology (CIRB), Paris (L.P.-F., A.E.); Department of Neurology, Yale University School of Medicine, New Haven, CT (J.-L.T.); Institut du Cerveau et de la Moelle, Inserm, Université Pierre et Marie Curie, Paris, France (J.-L.T.); Sorbonne Universités, UPMC Universités Paris 06, INSERM, UMR-S968, CNRS, UMR-7210, Institut de la Vision, France (A.C.); Departments of Cell Biology and Biomedical Engineering, Yale University, New Haven, CT (M.A.S.); and Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, CT (A.E.).
Background:
Sprouting angiogenesis is a key process driving blood vessel growth in ischemic tissues and an important drug target in a number of diseases, including wet macular degeneration and wound healing. Endothelial cells forming the sprout must develop front-rear polarity to allow sprout extension. The adaptor proteins Nck1 and 2 are known regulators of cytoskeletal dynamics and polarity, but their function in angiogenesis is poorly understood. Here, we show that the Nck adaptors are required for endothelial cell front-rear polarity and migration downstream of the angiogenic growth factors VEGF-A and Slit2.
Methods And Results:
Mice carrying inducible, endothelial-specific Nck1/2 deletions fail to develop front-rear polarized vessel sprouts and exhibit severe angiogenesis defects in the postnatal retina and during embryonic development. Inactivation of NCK1 and 2 inhibits polarity by preventing Cdc42 and Pak2 activation by VEGF-A and Slit2. Mechanistically, NCK binding to ROBO1 is required for both Slit2- and VEGF-induced front-rear polarity. Selective inhibition of polarized endothelial cell migration by targeting Nck1/2 prevents hypersprouting induced by Notch or Bmp signaling inhibition, and pathological ocular neovascularization and wound healing, as well.
Conclusions:
These data reveal a novel signal integration mechanism involving NCK1/2, ROBO1/2, and VEGFR2 that controls endothelial cell front-rear polarity during sprouting angiogenesis.
Insights
The adaptor proteins Nck1 and 2 are crucial for endothelial cell polarity and migration during sprouting angiogenesis. Their absence disrupts blood vessel formation in various conditions, highlighting their therapeutic potential.
Area of Science:
- Cell Biology
- Vascular Biology
- Molecular Medicine
Background:
- Sprouting angiogenesis drives blood vessel growth in ischemic tissues, a process vital for conditions like wet macular degeneration and wound healing.
- Endothelial cell polarity is essential for sprout extension during angiogenesis.
- The roles of adaptor proteins Nck1 and 2 in angiogenesis and endothelial cell polarity remain largely unknown.
Purpose of the Study:
- To investigate the function of Nck1 and 2 in endothelial cell polarity and migration during sprouting angiogenesis.
- To elucidate the molecular mechanisms by which Nck adaptors regulate angiogenesis downstream of VEGF-A and Slit2.
Main Methods:
- Utilized inducible, endothelial-specific Nck1/2 deletion in mice.
- Assessed angiogenesis defects in postnatal retinal and embryonic development models.
- Investigated the activation of Cdc42 and Pak2 in response to VEGF-A and Slit2.
- Examined the role of NCK binding to ROBO1 in polarity signaling.
Main Results:
- Endothelial-specific deletion of Nck1/2 severely impaired front-rear polarized vessel sprout formation and angiogenesis.
- Nck1/2 inactivation inhibited endothelial cell polarity by blocking Cdc42 and Pak2 activation by VEGF-A and Slit2.
- NCK binding to ROBO1 was essential for Slit2- and VEGF-induced polarity.
- Targeting Nck1/2 selectively inhibited polarized endothelial cell migration, preventing pathological neovascularization and improving wound healing.
Conclusions:
- Nck1/2 adaptors are critical regulators of endothelial cell front-rear polarity during sprouting angiogenesis.
- A novel signaling pathway involving NCK1/2, ROBO1/2, and VEGFR2 controls endothelial cell polarity in angiogenesis.
- Nck1/2 inhibition presents a potential therapeutic strategy for diseases involving pathological angiogenesis.
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