Targeting NCK-Mediated Endothelial Cell Front-Rear Polarity Inhibits Neovascularization

Abstract

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, as well as pathological ocular neovascularization and wound healing. 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.

Myc‐dependent endothelial proliferation is controlled by phosphotyrosine 1212 in VEGF receptor‐2

“…pY1212 has also been implicated in activation of the Roundabout (Robo) receptor by its ligand Slit. In this model, Robo and VEGFR2 are both required for binding Nck, leading to Cdc42 activation and EC front-rear polarity [19].…”

The Robo4 cytoplasmic domain is dispensable for vascular permeability and neovascularization

“…Mouse in vivo wound healing assay was described previously70. Eight-to-ten week old male mice were shaved and depilated on the back.…”

“…In vitro endothelial cell sprouting assays were performed as described70. Briefly, a layer of fibrin was made on the bottom of 24-well plates by mixing solubilized fibrinogen (10 mg ml −1 in EBM-2) with thrombin (final concentration 1 U).…”

Slit-Robo signaling

“…Many studies have linked Robo4 function to Slit (Enomoto et al, 2016;Fritz et al, 2015;Jones et al, 2009;Yu et al, 2014), although it was subsequently proposed that Robo4 does not bind Slits (Koch et al, 2011), with genetic evidence in knockout mice also failing to support a direct link between Robo4 and Slits (Rama et al, 2015). Endothelial cells also express Robo1/2 receptors and Slits, in particular Slit2, which favors angiogenesis by promoting endothelial cell motility and polarity (Dubrac et al, 2016;Rama et al, 2015). Given the overlapping expression pattern of Robo1/2 and Robo4, it is possible that Slit functions through receptor heterodimers without direct binding to Robo4.…”