Abstract 280: GIT1 Is Required for Pulmonary Vascular Development
The G-protein-coupled receptor (GPCR)-kinase interacting protein-1 (GIT1) is a multi-domain scaffold protein involved in multiple GPCR signal pathways including endocytosis, cell adhesion and migration. To determine the functions of GIT1 in vivo, we developed a traditional GIT1 knockout (KO) mouse. GIT1 KO mice exhibit ~60% mortality within 7 days post-birth. Pathologic examination showed that GIT1 KO mice display major defects in lung morphology including hemorrhage in parenchyma, increased alveolar spaces and impaired basement membrane formation. Micro-CT and fluorescence microangiography at day 5 demonstrated markedly reduced numbers of pulmonary blood vessels. Immunohistochemistry for vWF as a marker for endothelial cells (EC) confirmed the significant decrease in vessel density in lungs of GIT1 KO mice. The abnormalities in the GIT1 KO mice phenocopy the VEGF120 mouse and resemble bronchopulmonary dysplasia (BPD). BPD is a chronic lung disease that frequently develops in premature infants treated with ventilation and oxygen. BPD is characterized by decreased expression of VEGF and VEGFR2 and pulmonary microvascular deficiency. Because VEGF signaling involves activation of c-Src, PLC-γ, and ERK1/2, which we have shown are mediated by GIT1, we hypothesized that GIT1 is required for PLCγ activation by VEGF and lung microvasculature development. In cultured EC, knockdown of GIT1 by siRNA decreased phosphorylation of PLC-γ, while VEGF receptor 2 phosphorylation was not affected. Phosphorylation of downstream VEGF mediators PKC-β, MEK1/2 and ERK1/2 was significantly decreased. PLC-γ phosphorylation in lungs from GIT1 KO mice was also decreased.
These data support a critical role for GIT1 in pulmonary vascular development, by regulating VEGF induced PLC-γ activation. This mechanism may have relevance for clinical syndromes such as BPD.