Microtubule depolymerization rapidly collapses capillary tube networks in vitro and angiogenic vessels in vivo through the small GTPase Rho

Abstract

Maintenance of endothelial cell tube integrity is dependent on an intact cytoskeleton. We present data indicating that rapid collapse of endothelial tubular networks in vitro occurs in a dose-dependent manner after administration of microtubule-depolymerizing reagents but not after actin depolymerization. Pretreatment of endothelial cell networks with C3 exoenzyme or recombinant adenoviruses expressing dominant negative RhoA resulted in complete blockade of tube collapse, indicating a role for RhoA in these events. Microtubule depolymerization also resulted in activation of RhoA, whereas increased expression of constitutively active RhoA induced cell rounding and apoptosis of endothelial cells. Furthermore, following treatment with the chemotherapeutic agent vinblastine, rapid capillary tube network collapse occurred followed by endothelial cell apoptosis. Vinblastine, but not control agents, induced cleavage of procaspase-3, procaspase-9, and procaspase-8, along with the known caspase targets p21-activated kinase-2 and gelsolin, indicating that tube collapse caused a defined apoptotic response. Using a model of vascular endothelial growth factor-stimulated angiogenesis in vivo, vinblastine treatment also resulted in collapse and apoptosis of angiogenic blood vessels. Apoptotic endothelial cells stained strongly for cleaved caspase-3, and terminal dUTP nick-end labeling staining revealed fragmented nuclei in vinblastine-treated but not control angiogenic areas. Together, these findings indicate that microtubule-depolymerizing agents directly induce endothelial network collapse in vitro and in vivo leading to endothelial cell apoptosis in a manner dependent on the small GTPase, RhoA. In addition, these findings reveal a novel function for microtubule disrupting chemotherapeutic agents, namely their ability to rapidly collapse newly formed angiogenic vessels, which may contribute to their effectiveness in limiting angiogenesis and tumor growth.