RhoA is a small guanosine triphosphate hydrolase (GTPase) belonging to the Ras homology (Rho) family. The Rho family of GTPases comprise at least 23 members (25,26), which serve as key regulators of extracellular stimulus-mediated signaling networks involved in a diversity of cellular processes including motility, mitosis, proliferation and apoptosis (27). RhoA promotes actin stress fiber formation and focal adhesion assembly.

Rho GTPases function as molecular switches, cycling between an active GTP-bound conformation and an inactive guanosine diphosphate (GDP)-bound conformation (28,29). When binding with GTP, they interact with downstream effectors to propagate signal transduction (30). Intrinsic phosphatase activity hydrolyzes GTP to GDP, deactivating RhoA function, and this process is accelerated by interaction with GTPase-activating proteins. Conversely, interaction with guanine-nucleotide exchange factors facilitates the exchange of GDP to GTP, which restores the activation of RhoA. The relative affinity difference of the effector molecules between the GTP- and GDP-bound states of the Rho GTPase can be as much as 100-fold, resulting in a highly-specific interaction only with the GTP-bound activated state (Fig. 1). In addition, Rho proteins are also regulated by guanine nucleotide dissociation inhibitors (GDIs), which can inhibit both the exchange of GDP to GTP and the hydrolysis of bound GTP. In the majority of cases, Rho proteins are post-translationally modified at their C-termini by prenylation of a conserved cysteine, and this facilitates their attachment to cell membranes.

Rho kinase (ROCK) is a serine/threonine kinase with a molecular mass of ~160 kDa, which has been shown to be the principle downstream target of RhoA. There are two ROCK isoforms: ROCK1 (ROCKβ or p160 ROCK) and ROCK2 (ROCKα or Rho kinase). ROCK1 and ROCK2 show an overall homology of 65% in their amino acid sequence and 92% in their kinase domains. Both ROCK1 and ROCK2 are expressed in vascular endothelial cells (ECs) (31,32).

When bound and activated by RhoA, ROCK translocates from the cytoplasm to the cell membrane, where it increases phosphorylation of the myosin light chain (MLC) of myosin II. This is achieved either by direct phosphorylation, or by phosphorylation of the regulatory myosin-binding subunit of myosin phosphatase (also known as the phosphatase-targeting subunit), which inhibits the phosphatase activity of this molecule (33). This, in turn, enhances actin binding and the actin-induced adenosine triphosphatase activity of myosin, facilitating the interaction of myosin with F-actin, and ultimately cell contractility. ROCK proteins can also phosphorylate cofilin indirectly via LIM kinase, and this facilitates the organization of F-actin into stress fibers and re-arrangement of the actin cytoskeleton (30).

In eukaryotes, organization and reorganization of the cytoskeleton underpins cellular morphology and motility. The actin cytoskeleton is composed of actin filaments and numerous specialized actin-binding proteins. Actin filaments are created by the simple polymerization of actin monomers, regulated dynamically by numerous upstream signaling pathways, notably Rho GTPases (30). A coordinated regulation of the actin network is essential to produce directed cell movement.

During cell movement, RhoA is active at the trailing edge of the cell to promote retraction, while Rac, another member of the Rho family, is active at the leading edge, promoting protrusion. Active RhoA has also been shown to localize at the leading edge of migrating cells (34–36), indicating that RhoA not only acts in retraction, but also regulates protrusion at the front of the cell. Notably, increases in RhoA activity were found to be correlated with increases in protrusion rates, and were synchronous with cell-edge advancement (36). These data highlight the important role of RhoA in cell movement.

Angiogenesis is a five-step process involving a complex series of events. Firstly, an increase in the permeability of the basement membrane allows a new capillary to sprout. Next, ECs activated by angiogenic factors migrate through the basement membrane into the extracellular matrix, towards the angiogenic stimulus. The leading front of migrating cells is driven by enhanced proliferation of ECs. This is then followed by re-organization of ECs to form tubules with a central lumen, together with the recruitment of periendothelial cells (pericytes) and vascular smooth muscle cells for new capillary stabilization (37). The RhoA/ROCK pathway plays a role in each of these key steps (Fig. 2).

The endothelium is a semi-permeable barrier that lines the vasculature, comprising ECs that are connected to each other by interendothelial junctions, consisting of protein complexes organized as tight junctions and adherent junctions. The latter are in the majority (38), and are composed of vascular endothelial (VE) cadherin that associates homotypically with VE-cadherin on adjacent cells. VE-cadherin binds to the actin cytoskeleton. Actin-mediated EC contraction occurs as a result of MLC phosphorylation, and this can cause dysfunction of the endothelial barrier by inducing the formation of small gaps between neighboring cells (39). RhoA, through its downstream effector ROCK, plays a role in endothelial barrier dysfunction by potentiating MLC phosphorylation via inhibition of MLC phosphatase activity. Studies have also confirmed that RhoA contributes to VEGF-induced hyperpermeability in the endothelium (40).

The formation of stress fibers and cellular contraction is essential for EC migration, and these processes are mediated by Rho GTPases (41). van Nieuw Amerongen et al demonstrated in vitro that VEGF induces the activation of RhoA and this increase in RhoA activity is necessary for VEGF-induced reorganization of the F-actin cytoskeleton. This process can be inhibited by transfection of ECs with a RhoA dominant-negative mutant vector or by a RhoA inhibitor C3 (42). Zhao et al showed that increased expression of RhoA in human umbilical vein ECs significantly enhanced cytoskeletal reorganization of transfected cells, cell migration and angiogenic capacity, which suggests that RhoA plays a key part in these processes in vitro (43).

Several lines of evidence suggest that Rho proteins play an important role in normal and cancerous cell growth processes, including G1 phase cell cycle progression and mitogenesis (44). Cytokinesis is a step in mitogenesis which is critical within the cell cycle. In eukaryotic cells, cytokinesis requires an actin and myosin contractile ring, which constricts and cleaves the cell, forming two daughter cells. Inhibition of Rho GTPases prevents the assembly of this contractile ring in a variety of mammalian cells. Expression of constitutively activated Rho GTPases also blocks cytokinesis, suggesting that cycling between the active and inactive forms is required for its function (45).

The role of RhoA signaling in cell survival has been evaluated in several non-EC cell types. Results showed that inhibition of Rho signaling leads to apoptosis via alterations in cell adhesion and the induction of p53 and other pro-apoptotic proteins, or via ceramide upregulation leading to caspase cleavage and subsequent activation (46,47). Studies have shown that the ROCK inhibitors, fasudil and Y-27632, not only inhibit VEGF-induced cell proliferation, but also reverse the protective effect of VEGF on apoptosis, which results in a decrease in viability of VEGF-stimulated ECs (48,49). Data obtained with these inhibitors have revealed the important role of the RhoA/ROCK pathway in EC proliferation and cell viability.

Cultured ECs can undergo marked changes in shape and tube formation that closely imitate pre-capillary cord formation in vivo (50). In vitro angiogenesis assays found that the mean tube length of the capillary-like tubular structures formed by ECs was reduced by transfection of a RhoA dominant-negative mutant vector, the RhoA inhibitor C3, or the ROCK inhibitor Y-27632 (42). In another study, Zhao et al demonstrated that overexpression of RhoA increased the tube length in transfected ECs (43).