Abstract 20131: G Protein Coupled Receptor Kinase 2 Interacting Protein 1 is a Novel Mediator Required for Retina Vasculature Development Through Notch Signaling
G-protein-coupled receptor-kinase-2 interacting protein 1 (GIT1) is a scaffold protein. Our previous data showed GIT1 has a key role of in pulmonary vascular development by regulating VEGF mediated signaling. The Notch family regulates numerous cell fate/lineage decisions. Recently it has been shown that Notch1ligand, delta like 4(Dll4) induced by VEGF negatively regulates sprouting angiogenesis during retina vasculature development. Thus, we hypothesize that GIT1 is a novel mediator required for retina vasculature development through VEGF-Notch1-Dll4 pathway. As we anticipated, GIT1 KO mice demonstrated striking retina vascular phenotypes which include an obvious decrease in vessel length, endothelial tip cell numbers and filopodia extensions. Moreover, endothelial cell specific GIT1 KO mice displayed a similar phenotype. Ex vivo angiogenesis assay further confirmed that diminished GIT1 inhibited vessel sprouting. Upon VEGF stimulation or ligand binding, Notch1 is proteolytically cleaved by γ-secretase to generate the Notch intracellular domain (N1-ICD). N1-ICD translocates to the nucleus to stimulate target gene expression and promote angiogenesis. In turn, Notch1 activated by VEGF triggers the feedback loop which inhibits the response to VEGF through upregulation of Dll4. Surprisingly, we found GIT1 siRNA treatment significantly accelerated the cleavage of Notch1 induced by VEGF. Dll4 and downstream target gene hairy/enhancer-of-split1 (Hey1) expression was enhanced by deletion of GIT1. In vivo, Dll4 and Hey1expression in retinas from GIT1 KO mice were also increased compared to WT mice. To determine the mechanisms of GIT1 on Notch1-Dll4 signaling, γ-secretase activity was assayed. VEGF time dependently increased γ-secretase activity, whereas loss of GIT1 hastened the activation of γ-secretase. All these data imply that GIT1 acts as a “ switch off ” for Notch1 signaling. Inhibition of GIT1 induces the “switch on” of Notch1 pathway and its feedback loops, which subsequently inhibits the sprouting angiogenesis through Dll4. In conclusion, this report provides novel evidence of a previously unrecognized contribution of GIT1 to sprouting angiogenesis through VEGF-Notch1-Dll4 signaling.
- © 2010 by American Heart Association, Inc.