Overexpression of p27(Kip1) by doxycycline-regulated adenoviral vectors inhibits endothelial cell proliferation and migration and impairs angiogenesis

Formation of new blood vessels in the adult animal (i.e., angiogenesis) is an important event for tissue repair and for tumor growth and metastasis. A ngiogenesis involves the migration and proliferation of endothelial cells. We have investigated the role of the growth suppressor p27(Kip1) (p27) on e ndothelial cell function in vitro and angiogenesis in vivo. We have generat ed Ad-TetON, a replication-deficient adenovirus that constitutively express es the reverse tet-responsive transcriptional activator, and Ad-TRE-p27, wh ich drives expression of p27 under the control of the tet response element. Western blot analysis demonstrated doxycycline-dependent overexpression of p27 in human umbilical vein endothelial cells (HUVECs) coinfected with Ad- TetON and Ad-TRE-p27, which resulted in a marked inhibition of DNA replicat ion and cell migration in vitro. Inducible overexpression of p27 in culture d HUVECs inhibited the formation of tubelike structures and, when applied i n a murine model of hind limb ischemia, reduced hind limb blood flow recove ry and capillary density. These findings thus underscore a novel role of p2 7 in regulating endothelial cell migration in vitro and angiogenesis in viv o, suggesting a novel anti-angiogenic therapy based on inducible p27 overex pression.