Active RhoA Exerts an Inhibitory Effect on the Homeostasis and Angiogenic Capacity of Human Endothelial Cells

BACKGROUND: The small GTPase RhoA (Ras homolog gene family, member A) regulates a variety of cellular processes, including cell motility, proliferation, survival, and permeability. In addition, there are reports indicating that RhoA‐ROCK (rho associated coiled‐coil containing protein kinase) activation is essential for VEGF (vascular endothelial growth factor)‐mediated angiogenesis, whereas other work suggests VEGF‐antagonistic effects of the RhoA‐ROCK axis. METHODS AND RESULTS: To elucidate this issue, we examined human umbilical vein endothelial cells and human coronary artery endothelial cells after stable overexpression (lentiviral transduction) of constitutively active (G14V/Q63L), dominant‐negative (T19N), or wild‐type RhoA using a series of in vitro angiogenesis assays (proliferation, migration, tube formation, angiogenic sprouting, endothelial cell viability) and a human umbilical vein endothelial cells xenograft assay in immune‐incompetent NOD scid gamma mice in vivo. Here, we report that expression of active and wild‐type RhoA but not dominant‐negative RhoA significantly inhibited endothelial cell proliferation, migration, tube formation, and angiogenic sprouting in vitro. Moreover, active RhoA increased endothelial cell death in vitro and decreased human umbilical vein endothelial cell‐related angiogenesis in vivo. Inhibition of RhoA by C3 transferase antagonized the inhibitory effects of RhoA and strongly enhanced VEGF‐induced angiogenic sprouting in control‐treated cells. In contrast, inhibition of RhoA effectors ROCK1/2 and LIMK1/2 (LIM domain kinase 1/2) did not significantly affect RhoA‐related effects, but increased angiogenic sprouting and migration of control‐treated cells. In agreement with these data, VEGF did not activate RhoA in human umbilical vein endothelial cells as measured by a Förster resonance energy transfer–based biosensor. Furthermore, global transcriptome and subsequent bioinformatic gene ontology enrichment analyses revealed that constitutively active RhoA induced a differentially expressed gene pattern that was enriched for gene ontology biological process terms associated with mitotic nuclear division, cell proliferation, cell motility, and cell adhesion, which included a significant decrease in VEGFR‐2 (vascular endothelial growth factor receptor 2) and NOS3 (nitric oxide synthase 3) expression. CONCLUSIONS: Our data demonstrate that increased RhoA activity has the potential to trigger endothelial dysfunction and antiangiogenic effects independently of its well‐characterized downstream effectors ROCK and LIMK.