RhoA/ROCK signaling is essential for multiple aspects of VEGF‐mediated angiogenesis

Abstract

The small GTPase RhoA and its downstream effectors, ROCK1 and ROCK2, regulate a number of cellular processes, including cell motility, proliferation, survival, and permeability. Pharmacological inhibitors of the Rho pathway reportedly block angiogenesis; however, the molecular details of this inhibition are largely unknown. We demonstrate that vascular endothelial growth factor-A (VEGF) rapidly induces RhoA activation in endothelial cells (ECs). Moreover, the pharmacological inhibition of ROCK1/2 using 10 microM Y-27632 (the IC(50) for this compound in ECs) strongly disrupts vasculogenesis in pluripotent embryonic stem cell cultures, VEGF-mediated regenerative angiogenesis in ex vivo retinal explants, and VEGF-mediated in vitro EC tube formation. Furthermore, using small interfering RNA knockdown and mouse heterozygote knockouts of ROCK1 and ROCK2, we provide data indicating that VEGF-driven angiogenesis is largely mediated through ROCK2. These data demonstrate that Rho/ROCK signaling is an important mediator in a number of angiogenic processes, including EC migration, survival, and cell permeability, and suggest that Rho/ROCK inhibition may prove useful for the treatment of angiogenesis-related disorders.

Rock1 & 2 Perform Overlapping and Unique Roles in Angiogenesis and Angiosarcoma Tumor Progression

“…We have previously demonstrated that ablation of ROCK1 & 2 kinase activity with the non-selective ROCK1 & 2 pharmacological inhibitor Y27632 resulted in disrupted angiogenesis [7]. To determine the contribution of the individual ROCK paralogs to capillary network formation, the endothelial cell panel stably overexpressing non-targeting, ROCK1, or ROCK2 shRNA plasmids was subjected to matrigel network formation assays.…”

“…MS1 mouse pancreatic endothelial cells (ATCC# CRL-2279), SVR mouse engineered angiosarcoma cells (ATCC# CRL-2280), and B16F1 mouse melanoma cells (ATCC# CRL-6323) were maintained in Dulbecco’s modified Eagle’s media (DMEM) supplemented with 10% fetal bovine serum (FBS), 80 U/ml penicillin, and 50 µg/ml streptomycin C. Cells were treated as indicated with the following concentrations: human recombinant VEGF 165 (VEGF) (2.5 ng/ml) or Y27632 (10 mM) as previously described [7]. shRNA vectors (SABiosciences) (Table 1 ) were transfected using Lipofectamine 2000, and cell pools were stably selected with hygromycin ( MS1 cells ) or puromycin ( SVR cells ).…”

“…ROCK1(-/-) mice result in lethality soon after birth, displaying failure of eyelid and ventral body wall closure [3], while ROCK2(-/-) mice experience embryonic lethality due to interuterine growth retardation and placental dysfunction [4]. Viable fertile litters have been reported for ROCK1(+/-) and ROCK2(+/-) mice, however ROCK1(+/-) mice exhibit increased resistance to perivascular fibrosis and reduced vascular injury-induced neointima formation [5, 6], while ROCK2(+/-) mice display decreased platelet endothelial cell adhesion molecule staining of endothelial cells in the lung [7]. Only a handful of recent reports have utilized RNAi technology to singularly disrupt each ROCK paralog in vitro , demonstrating unique roles for each protein in the control of actin-cytoskeleton dynamics and cell morphogenesis, migration, cell fate decisions, and extracellular matrix assembly [7-13].…”

“…Viable fertile litters have been reported for ROCK1(+/-) and ROCK2(+/-) mice, however ROCK1(+/-) mice exhibit increased resistance to perivascular fibrosis and reduced vascular injury-induced neointima formation [5, 6], while ROCK2(+/-) mice display decreased platelet endothelial cell adhesion molecule staining of endothelial cells in the lung [7]. Only a handful of recent reports have utilized RNAi technology to singularly disrupt each ROCK paralog in vitro , demonstrating unique roles for each protein in the control of actin-cytoskeleton dynamics and cell morphogenesis, migration, cell fate decisions, and extracellular matrix assembly [7-13]. These data collectively suggest that ROCK1 and 2 paralogs perform, at least to some degree, unique biological roles in cell function.…”

“…Using in vivo and in vitro angiogenic assays, several labs have reported that disruption of RhoA/ROCK signaling inhibits vascular endothelial growth factor (VEGF)-mediated endothelial cell activation [7, 14-19]. Moreover, tumor derived endothelial cells display an enhanced ability to organize into capillary networks, correlating with a constitutively high level of RhoA/ROCK signaling [20].…”