Angiogenesis, the process by which new blood vessels are formed from preexisting vasculature, is critical for vascular remodeling during development and contributes to the pathogenesis of diseases such as cancer. Two critical steps in this process are endothelial cell migration and assembly into new tubules. Over the last decade, a diverse array of molecular regulators that participate in the process of angiogenesis has been identified (4
). The Eph family of receptor tyrosine kinases is one such family of angiogenic regulators that plays a prominent role in endothelial cell assembly and migration.
The Eph receptors belong to the largest family of receptor tyrosine kinases in the genome, with 16 receptors and 9 ligands identified to date in vertebrates (28
). Based on binding specificity and structural properties, the Eph receptors are divided into two subclasses, class A and class B (23
). In general, EphA receptors bind to glycosylphosphatidylinositol-linked ephrin-A ligands, while EphB receptors bind to transmembrane ephrin-B ligands. Gene targeting studies have established several class B Eph family members as key regulators of embryonic vascular development (2
). In contrast, class A Eph receptors have been shown to regulate postnatal angiogenesis in adults. Ephrin-A1 stimulates endothelial cell migration and assembly in culture (15
) and induces corneal angiogenesis in vivo (37
). More recently, Eph receptors have been detected in tumor blood vessel endothelial cells (reviewed in references 8
). Inhibition of class A Eph receptor signaling by soluble EphA2-Fc or EphA3-Fc receptors decreased tumor volume, tumor angiogenesis, and metastatic progression in vivo (6
). A main target of soluble EphA receptors appears to be EphA2, as EphA2-deficient endothelial cells fail to migrate and assemble in vitro (7
), and loss of EphA2 receptor resulted in impaired tumor growth and metastasis in vivo (10
). These data support the crucial role for Eph receptor-mediated regulation of angiogenesis.
Investigation of ephrin/Eph receptor-mediated signal transduction mechanisms that regulate cellular responses in various cell types has been centered on Rho-family GTPases (33
). In vascular smooth muscle cells, for example, the EphA4 receptor stimulates RhoA activity via direct interaction with Vsm-RhoGEF (35
), while ephrin-A1 stimulation inhibits Rac1 and p21-activated kinase (PAK) activity (17
). In endothelial cells, however, EphA2 receptor-mediated cell migration is dependent on Rac1 GTPase activation (7
). Ephrin-A1 stimulation induces activation of the Rac1 GTPase, and a dominant negative N17 Rac1 mutant inhibits ephrin-A1-induced endothelial cell motility. Rac1 activity also appears to be regulated by phosphatidylinositol 3 kinase (PI3K). PI3K-specific inhibitors, wortmannin, LY294002, or a dominant negative p85 subunit of PI3K, block ephrin-A1-induced Rac1 activation and endothelial cell migration. These data suggest that the EphA2 receptor controls endothelial cell motility by regulating Rac1 GTPase activity.
The molecular mechanism by which the EphA2 receptor regulates the activity of Rac1 GTPase in endothelial cells remains to be elucidated. The Vav family of guanine nucleotide exchange factors (GEFs), which includes Vav1, Vav2, and Vav3, has been shown to modulate activity of Rho, Rac, and/or Cdc42 to elicit changes in cytoskeletal organization (27
). In addition, Vav proteins can interact with the PI3K lipid product phosphatidylinositol-3,4,5-triphosphate in activation of Rac1 (16
). While the majority of studies on these proteins have focused on functions in the immune system (45
), Vav2 and Vav3 display a broader tissue expression profile and therefore likely regulate cytoskeletal dynamics in other cells types (27
). Vav2 and Vav3 are expressed in heart and other highly vascularized organs, including placenta, lung, and kidney (44
). As EphA2-mediated Rac1 activation in endothelial cells is PI3K-dependent (7
), these data suggest that Vav proteins may link the EphA2 receptor to Rac1 directly and/or through PI3K.
Through a yeast two-hybrid screen, we identified the Vav3 GEF as a binding partner of EphA2. In this study, we report that both Vav3 and Vav2 GEFs are recruited to phosphorylated EphA2 receptors in mammalian cells. Endothelial cells deficient in both Vav2 and Vav3 exhibit impaired activation of Rac1 GTPase and endothelial cell migration and assembly in response to ephrin-A1 ligand. In addition, loss of both Vav2 and Vav3 inhibits ephrin-A1-induced angiogenesis in vivo. We propose that regulation of Rac1 GTPase signaling by modulation of Vav2/3 activity may underlie endothelial responses to ephrin-A1.