Role of GTPases in control of microvascular permeability.
ABSTRACT Inflammatory mediators increase vascular permeability primarily by formation of intercellular gaps between endothelial cells of post-capillary venules. Under these conditions, endothelial cell-cell contacts such as adherens and tight junctions open to allow paracellular fluid passage. Small guanosine triphosphatases (GTPases) from the ras superfamily, primarily Rho GTPases (RhoA, Rac1, Cdc42) or Rap1 are known to regulate cell adhesion, in part by reorganization of the junction-associated cortical actin cytoskeleton. In this review, we will discuss the role of small GTPases for the maintenance of microvascular barrier functions under resting conditions as well as under conditions of increased permeability and their involvement in signalling pathways downstream of both barrier-stabilizing and inflammatory mediators. Rac1 and Cdc42 are the main GTPases required for barrier maintenance and stabilization, whereas RhoA negatively regulates barrier properties under both resting and inflammatory conditions. For Rac1 and RhoA, contrary functions under certain conditions have also been described. However, Rac1-mediated barrier destabilization in microvascular endothelium appears to be largely restricted to conditions of enhanced endothelial cell migration and thus to be more closely related to angiogenesis rather than to inflammation. Recent studies revealed that cAMP signalling, which is well known to be barrier protective, enhances barrier functions in part via Rap1-mediated activation of Rac1 and Cdc42 as well as by inhibition of RhoA. Moreover, barrier-stabilizing mediators directly activate Rac1 and Cdc42 or increase cAMP levels. On the other hand, several barrier-disruptive components appear to increase permeability by reduced formation of cAMP, leading to both inactivation of Rac1 and activation of RhoA.
- SourceAvailable from: Shahram Eisa-Beygi[Show abstract] [Hide abstract]
ABSTRACT: Activation of the RhoA/Rho-kinase (ROCK) pathway induces endothelial barrier dysfunction and increased vascular permeability, which is a hallmark of various life- threatening vascular pathologies. Therapeutic approaches aimed at inhibiting the RhoA/ROCK pathway have proven effective in the attenuation of vascular leakage observed in animal models of endotoxin-induced lung injury/sepsis, edema, autoimmune disorders, and stroke. These findings suggest that treatments targeting the ROCK pathway might be of benefit in the management of the Ebola virus disease (EVD), which is characterized by severe vascular leak, likely involving pro-inflammatory cytokines, such as tumor necrosis factor-alpha, released from virus-infected macrophages. In this paper, we review evidence from in vivo and in vitro models of vascular leakage, suggesting that the RhoA/ROCK pathway is an important therapeutic target for the reversal of the vascular permeability defects associated with EVD. Future studies should explore the efficacy of pharmacological inhibition of RhoA/ROCK pathway on reversing the endothelial barrier dysfunction in animal models of EVD and other hemorrhagic fever virus infections as part of an adjunctive therapy. Such experimental studies should focus, in particular, on the small molecule fasudil (HA-1077), a derivative of isoquinoline, which is a safe and clinically approved inhibitor of ROCK, making it an excellent candidate in this context.Antiviral Research 12/2014; 114. DOI:10.1016/j.antiviral.2014.12.005 · 3.43 Impact Factor
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ABSTRACT: This study is to examine whether the activation of Rho kinase (ROCK) accounts for hemoglobin (Hb)-induced disruption of blood-brain barrier (BBB) after the occurrence of intracerebral hemorrhage. A model of intracerebral injection of Hb was established in rats. Changes in the levels of mRNA of RhoA, ROCK2 and matrix metalloproteinase-9 (MMP-9) were measured using quantitative real-time polymerase chain reaction. Protein expression of RhoA, ROCK2, claudin-5 and MMP-9, as well as ROCK activity, were determined using Western blotting. Immunohistochemical assay was performed to visualize the expression of RhoA, ROCK2, claudin-5 and MMP-9 in endothelial cells. Hb injection produced a significant increase in BBB permeability and water content in the brain. Significant reduction of claudin-5 expression was detected by Western blotting and immunofluorescence in Hb group. The levels of RhoA and ROCK2 were significantly up-regulated from 6 h to 12 h after Hb injection and were concomitant with the increase in ROCK activity. Immunofluorescence double staining showed enhanced p-myosin light chain immunoreactivity but diminished claudin-5 staining in endothelial cells. Significant up-regulation of MMP-9 expression was detected after Hb injection, and statistical analyses further confirmed a positive correlation of MMP-9 expression with ROCK activity. The results showed that ROCK was activated in endothelial cells by Hb. This may account for the early disruption of the BBB via up-regulation of p-myosin light chain expression and aggravation of injuries to TJ proteins. The activation of ROCK may also increase MMP-9 expression, thereby leading to further BBB disruption.
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ABSTRACT: Vascular endothelial cadherin (VE-cadherin) mediates homophylic adhesion between endothelial cells and is an important regulator of angiogenesis, blood vessel permeability and leukocyte trafficking. Rac1, a member of the Rho family of GTPases, controls VE-cadherin adhesion by acting downstream of several growth factors, including angiopoietin-1 and vascular endothelial growth factor (VEGF). Here we show that UTP-induced activation of the Gq protein-coupled P2Y2 nucleotide receptor (P2Y2R) in human coronary artery endothelial cells (HCAECs) activated Rac1 and caused a transient complex to form between P2Y2R, VE-cadherin and VEGF receptor-2 (VEGFR-2). Knockdown of VE-cadherin expression with siRNA did not affect UTP-induced activation of extracellular signal-regulated kinases 1/2 (ERK1/2) but led to a loss of UTP-induced Rac1 activation and tyrosine phosphorylation of p120 catenin, a cytoplasmic protein known to interact with VE-cadherin. Activation of the P2Y2R by UTP also caused a prolonged interaction between p120 catenin and vav2 (a guanine nucleotide exchange factor for Rac) that correlated with the kinetics of UTP-induced tyrosine phosphorylation of p120 catenin and VE-cadherin. Inhibitors of VEGFR-2 (SU1498) or Src (PP2) significantly diminished UTP-induced Rac1 activation, tyrosine phosphorylation of p120 catenin and VE-cadherin, and association of the P2Y2R with VE-cadherin and p120 catenin with vav2. These findings suggest that the P2Y2R uses Src and VEGFR-2 to mediate association of the P2Y2R with VE-cadherin complexes in endothelial adherens junctions to activate Rac1.