Abstract 5439: Scaffolding Docking Protein Gab1 Is Essential for HGF-Mediated Endothelial Signaling and Angiogenesis after Hind-Limb Ischemia
Various receptor tyrosine kinases (RTKs) have essential roles for angiogenesis. Docking protein Gab1 coordinates activation of both ERK1/2 and AKT downstream of various RTKs. However, the role of Gab1 in angiogenesis remains elusive. Here, we aim to elucidate the role of Gab1 in angiogenesis through creating endothelium-specific Gab1 conditional knockout mice (Gab1ECKO). Gab1ECKO were viable and showed no obvious developmental defects. To decipher the functional role of Gab1 in the endothelium, we created a murine model of operatively induced hindlimb ischemia. Angiogenesis in the ischemic limb was significantly impaired in Gab1ECKO versus control evaluated by either doppler flow intensity or capillary density measurement. Intriguingly, necrosis in the operated limb was observed in all of Gab1ECKO, but not in control during 3 weeks observation. To identify the angiogenic factor utilizing Gab1 in the endothelium, we stimulated human umbilical vein endothelial cells (HUVECs) with vascular endothelial growth factor, hepatocyte growth factor (HGF), or fibroblast growth factor 2. Among these, HGF induced the most prominent tyrosine-phosphorylation of Gab1, leading to strong association of Gab1 with tyrosine phosphatase SHP2 and PI3-kinase p85 subunit. Next, we examined the effect of adenovirus-mediated overexpression of β-galactosidase (β-gal), wild-type Gab1 (Gab1WT), or mutated Gab1 which can’t bind with SHP2 (Gab1ΔSHP2) or p85 (Gab1Δp85). HGF-induced ERK1/2 activation was enhanced in the HUVECs expressing Gab1WT or Gab1Δp85, but abrogated in those expressing Gab1ΔSHP2, compared with control expressing β-gal. HGF-induced AKT activation was specifically attenuated in cells expressing Gab1Δp85, compared with other three groups. Furthermore, HGF-induced cell migration was enhanced in those expressing Gab1WT, but abrogated in those expressing Gab1ΔSHP2 or attenuated in those expressing Gab1Δp85. Consistent with these findings, intramuscular injection of HGF expression plasmid improved either doppler flow intensity or capillary density score in control mice, but not in Gab1ECKO. Collectively, these data indicate that Gab1 is essential for HGF-dependent endothelial signaling and angiogenesis after ischemia.