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Updated: Apr 18, 2026
Strategic Endothelial Cell Tube Formation Assay: Comparing Extracellular Matrix and Growth Factor Reduced Extracellular Matrix
Published on: August 14, 2016
Regulation of endothelial cell proliferation and vascular assembly through distinct mTORC2 signaling pathways
Shan Wang1, Katherine R Amato2, Wenqiang Song1
1Division of Rheumatology and Immunology, Department of Medicine, Vanderbilt University, Nashville, Tennessee, USA.
Abstract:
Mammalian target of rapamycin (mTOR) is a serine/threonine kinase that regulates a diverse array of cellular processes, including cell growth, survival, metabolism, and cytoskeleton dynamics. mTOR functions in two distinct complexes, mTORC1 and mTORC2, whose activities and substrate specificities are regulated by complex specific cofactors, including Raptor and Rictor, respectively. Little is known regarding the relative contribution of mTORC1 versus mTORC2 in vascular endothelial cells. Using mouse models of Raptor or Rictor gene targeting, we discovered that Rictor ablation inhibited vascular endothelial growth factor (VEGF)-induced endothelial cell proliferation and assembly in vitro and angiogenesis in vivo, whereas the loss of Raptor had only a modest effect on endothelial cells (ECs). Mechanistically, the loss of Rictor reduced the phosphorylation of AKT, protein kinase Cα (PKCα), and NDRG1 without affecting the mTORC1 pathway. In contrast, the loss of Raptor increased the phosphorylation of AKT despite inhibiting the phosphorylation of S6K1, a direct target of mTORC1. Reconstitution of Rictor-null cells with myristoylated AKT (Myr-AKT) rescued vascular assembly in Rictor-deficient endothelial cells, whereas PKCα rescued proliferation defects. Furthermore, tumor neovascularization in vivo was significantly decreased upon EC-specific Rictor deletion in mice. These data indicate that mTORC2 is a critical signaling node required for VEGF-mediated angiogenesis through the regulation of AKT and PKCα in vascular endothelial cells.
Insights
Mammalian target of rapamycin complex 2 (mTORC2) is crucial for blood vessel formation. Rictor deletion impaired vascular endothelial growth factor (VEGF)-induced angiogenesis by affecting AKT and PKCα signaling in endothelial cells.
Area of Science:
- Cell Biology
- Molecular Biology
- Vascular Biology
Background:
- Mammalian target of rapamycin (mTOR) is a key regulator of cellular processes, existing in two complexes: mTORC1 and mTORC2.
- The specific roles of mTORC1 versus mTORC2 in vascular endothelial cells (ECs) remain largely uncharacterized.
- Raptor and Rictor are key cofactors for mTORC1 and mTORC2, respectively.
Purpose of the Study:
- To elucidate the distinct roles of mTORC1 and mTORC2 in vascular endothelial cells.
- To investigate the contribution of Rictor and Raptor to vascular endothelial growth factor (VEGF)-induced angiogenesis.
Main Methods:
- Utilized mouse models with targeted deletion of Raptor or Rictor in endothelial cells.
- Assessed endothelial cell proliferation, assembly in vitro, and angiogenesis in vivo.
- Analyzed signaling pathways, including phosphorylation of AKT, PKCα, NDRG1, and S6K1.
- Performed rescue experiments using myristoylated AKT (Myr-AKT) and PKCα.
Main Results:
- Rictor deletion significantly inhibited VEGF-induced EC proliferation, assembly, and angiogenesis, while Raptor deletion had minimal impact.
- Loss of Rictor reduced AKT, PKCα, and NDRG1 phosphorylation but did not affect the mTORC1 pathway.
- Loss of Raptor increased AKT phosphorylation but inhibited S6K1 phosphorylation.
- Restoration of AKT or PKCα partially rescued Rictor-deficient EC defects.
- EC-specific Rictor deletion decreased tumor neovascularization in vivo.
Conclusions:
- mTORC2, specifically through Rictor, is essential for VEGF-mediated angiogenesis in vascular endothelial cells.
- mTORC2 regulates angiogenesis via the AKT and PKCα signaling pathways.
- mTORC2 plays a more critical role than mTORC1 in endothelial cell function and blood vessel development.
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