Cdc42 Regulates the Restoration of Endothelial Adherens Junctions and Permeability

doi:10.1016/j.tcm.2007.03.004

Trends in Cardiovascular Medicine

Volume 17, Issue 5, July 2007, Pages 151-156

https://doi.org/10.1016/j.tcm.2007.03.004 Get rights and content

The endothelial adherens junction (AJ) complex consisting of VE-cadherin and its associated catenins is a major determinant of fluid, solute, and plasma protein permeability of the vessel wall endothelial barrier. Impairment of endothelial barrier function contributes to cardiovascular diseases such as vascular inflammation and atherosclerosis. Adherens junctions disassemble in response to proinflammatory mediators, producing an increase in endothelial permeability; however, AJs also have the capacity to reassemble, leading to restoration of endothelial barrier function. Activation of Cdc42, a member of the Rho family of monomeric GTPases, is an essential signal regulating reannealing of AJs and reversal of the increase in endothelial permeability. The possibility of activating Cdc42 therapeutically represents a novel approach to prevent inflammatory diseases resulting from breakdown of the endothelial barrier. This review summarizes recent findings concerning the role of Cdc42 in restoring endothelial barrier integrity.

Section snippets

Structure of Endothelial Adherens Junctions

Adherens junctions are formed by Ca²⁺-dependent homotypic binding of the extracellular region of VE-cadherin to another VE-cadherin protein on the neighboring cell's plasma membrane (Figure 1A). The VE-cadherin interaction is regulated by the binding of intracellular domains of VE-cadherin to the catenin proteins (p120-catenin [p120ctn], β-catenin, and γ-catenin) (Lampugnani et al. 1995). The strength of intercellular adhesion mediated by VE-cadherin is dependent on the association of its

Role of Adherens Junctions in the Mechanism of Vascular Inflammation

Adherens junctions are responsible for the normal functioning of the endothelial barrier. Mice with deletions of either VE-cadherin or VE-cadherin's β-catenin-binding domain were embryonically lethal because of severely impaired vasculogenesis (Carmeliet et al. 1999). In adult mice, transduction of the cadherin mutant lacking the VE-cadherin extracellular domain (Broman et al. 2006), disruption of homotypic binding of cadherin proteins using anti–VE-cadherin antibody (Corada et al. 2002), and

Cdc42-Mediated Restoration of Endothelial Barrier Function

The Rho GTPase Cdc42 is particularly important in restoring endothelial junctional integrity. Studies showed that RhoA activation leads to formation of contractile stress fibers, whereas Cdc42 and, to a lesser extent, Rac1 coordinate the reorganization of actin filaments into membrane ruffles, filopodia, and lamellipodia (Hall 2005). Cdc42- and Rac1-induced actin cytoskeletal reorganization induces the apposition of AJs at the plasma membrane and promotes AJ reannealing. Both Cdc42 and Rac1

Mechanisms of Cdc42-Mediated Endothelial Adherens Junctional Repair

There is considerable speculation concerning the mechanisms of Cdc42-mediated reannealing of AJs. It is likely that repair of AJs requires the proper targeting of AJ proteins, VE-cadherin and catenins, and actin monomers at sites in the plasma membrane. Cdc42 is known to bind partitioning-defective protein 6 (Nishimura et al. 2005) and PKCζ (Plant et al. 2003), which may induce endothelial cell polarity during AJ repair. In addition, Cdc42, by interacting with vesicular trafficking proteins

Conclusions and Future Directions

Whereas the increase in endothelial permeability in response to humoral and inflammatory cells is important for host-tissue defense and tissue fluid balance, the failure to restore endothelial barrier function likely underlies the morbidity and mortality associated with vascular diseases such as ischemia-reperfusion injury, atherosclerosis, acute lung injury, septic shock, and metastasis. An understanding of the signals responsible for reversing the increase in endothelial permeability may

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Cdc42, a member of the Rho family of small GTPase, plays key roles in several cellular phenomena, such as cell growth, cell polarity, RNA processing, actin polymerization and intracellular trafficking [1–6]. Defects in Cdc42 regulations consequently lead to cancer, cardiovascular diseases, and neurodegenerative diseases [4,7–9]. In response to upstream external signals, guanine nucleotide exchange factors mediate the exchange of GDP for GTP in the nucleotide binding site to activate Cdc42 [10,11].
Cdc42 regulates pathways related to cell division. Dysregulation of Cdc42 can lead to cancer, cardiovascular diseases and neurodegenerative diseases. GTP induced activation mechanism plays an important role in the activity and biological functions of Cdc42. P-loop, Switch I and Switch II are critical regions modulating the enzymatic activity of Cdc42. We applied amide hydrogen/deuterium exchange coupled with liquid chromatography mass spectrometry (HDXMS) to investigate the dynamic changes of apo-Cdc42 after GDP, GTP and GMP-PCP binding. The natural substrate GTP induced significant decreases of deuteration in P-loop and Switch II, moderate changes of deuteration in Switch I and significant changes of deuteration in the α7 helix, a region far away from the active site. GTP binding induced similar effects on H/D exchange to its non-hydrolysable analog, GMP-PCP. HDXMS results indicate that GTP binding blocked the solvent accessibility in the active site leading to the decrease of H/D exchange rate surrounding the active site, and further triggered a conformational change resulting in the drastic decrease of H/D exchange rate at the remote α7 helix. Comparing the deuteration levels in three activation states of apo-Cdc42, Cdc42-GDP and Cdc42-GMP-PCP, the apo-Cdc42 has the most flexible structure, which can be stabilized by guanine nucleotide binding. The rates of H/D exchange of Cdc42-GDP are between the GMP-PCP-bound and the apo form, but more closely to the GMP-PCP-bound form. Our results show that the activation of Cdc42 is a process of conformational changes involved with P-loop, Switch II and α7 helix for structural stabilization.
Combined peri-ischemic administration of Bβ<inf>15-42</inf> in treating ischemia reperfusion injury of the mouse kidney
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The disruption of endothelial integrity is a crucial step for the development of vascular leakage and consequently ischemia-reperfusion injury (IRI). Regarding the molecular cell–cell interaction, the fibrinopeptide Bβ_15–42 prevents vascular leakage by stabilizing the inter-endothelial junctions via association with the vascular endothelial-cadherin. In a previous study we showed that a renoprotective effect in early IRI may be achieved by intravenous administration of Bβ_15–42 at the time of reperfusion. We now aimed to investigate whether additional pre-ischemic application of Bβ_15–42 could enhance this effect. Therefore C57BL/6 mice were subjected to 0.5 h bilateral renal ischemia followed by reperfusion. The animals were randomized into 6 groups (n = 6): two control groups treated with i.v. administration of NaCl at reperfusion for 0.5 h (NaCl 1 h) and 2.5 h (NaCl 3 h), two groups with Bβ_15–42 at reperfusion for 0.5 h (Bβ^rep 1 h) and 2.5 h (Bβ^rep 3 h), and two groups with administration of Bβ_15–42 immediately pre-ischemic as well as at reperfusion for 0.5 h (Bβ^peri 1 h) and 2.5 h (Bβ^peri 3 h). We found that both Bβ^rep and Bβ^peri mice displayed reduced early renal damage compared with NaCl treated mice. However, there was no further reduction of the IR damage through added pre-ischemic application of Bβ_15–42. Overall, we detected significantly reduced endothelial activation, lower tissue infiltration of neutrophils as well as lower tissue levels of neutrophil gelatinase-associated lipocalin (NGAL) in all mice treated with Bβ_15–42 compared to mice treated with NaCl. Our data confirm the renoprotective effect of Bβ_15–42 in the early therapeutic treatment of acute kidney injury due to ischemia and reperfusion. However, a combined pre-and post-ischemic administration of Bβ_15–42 appears to provide no additional benefit compared with a sole administration at reperfusion.
A global proteome approach in uric acid stimulated human aortic endothelial cells revealed regulation of multiple major cellular pathways
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Those proteins represent pathways involved in endothelial barrier function: (CDC42) [30], cell vitality and apoptosis prevention (ITB1) [31], and intracellular protein sorting, trafficking and signaling (RAB1A [32], GDIA [33]). Indeed, recent studies promote the targeting of CDC42 as a novel approach to prevent inflammatory processes resulting from endothelial barrier brake-down in cardiovascular disease [34]. Our proteomic data also allowed the identification of pathways based on peptide abundance using IPA.
Uric acid (UA) has been identified as one major risk factor for cardiovascular diseases. Lowering of serum UA levels improves endothelial function. The present study investigates for the first time concentration-dependent effects of UA on human aortic endothelial cells (HAEC) and the cellular pathways involved in global proteomic analysis.
The concentration dependent effects of UA on HAEC were investigated by nanoLC–MS/MS and ingenuity pathway analysis to reveal putative cellular pathways. For verification of the identified pathways the abundance or activity of key proteins was measured using ELISA or Western blotting. NO production was quantified by confocal laser microscopy.
We identified ubiquitin–proteasome system (UPS) and eIF4 signaling as the major pathways regulated by UA. K-means clustering analysis revealed 11 additional pathways, of which NO, superoxide signaling and hypoxia were further analyzed. A complex regulatory network was detected demonstrating that 500 μmol/L UA, which is well above the concentration regarded as pathological in clinical settings, led to diminishing of NO bioavailability. In addition a UA-dependent downregulation of eIF4, an upregulation of UPS and an increase in HIF-1α were detected.
Here we show for the first time, that increasing UA levels activate different sets of proteins representing specific cellular pathways important for endothelial function. This indicates that UA may alter far more pathways in HAEC than previously assumed. This regulation occurs in a complex manner depending on UA concentration. Further studies in knockout and overexpression models of the identified proteins are necessary to prove the correlation with endothelial dysfunction.
Src-induced tyrosine phosphorylation of VE-cadherin is not sufficient to decrease barrier function of endothelial monolayers
2010, Journal of Biological Chemistry
Citation Excerpt :
However, we did not see an increase in VE-cadherin endocytosis in our fluorescence experiments, even after caSrc overexpression, suggesting that this pathway may not be essential in this model. Also, it is possible that other GTPases regulating permeability may be differentially regulated (see Ref. 34 and references therein). Surprisingly, we also did not find a change in the association of VE-cadherin to either β-catenin or p120.
Activation of Src family kinases (SFK) and the subsequent phosphorylation of VE-cadherin have been proposed as major regulatory steps leading to increases in vascular permeability in response to inflammatory mediators and growth factors. To investigate Src signaling in the absence of parallel signaling pathways initiated by growth factors or inflammatory mediators, we activated Src and SFKs by expression of dominant negative Csk, expression of constitutively active Src, or knockdown of Csk. Activation of SFK by overexpression of dominant negative Csk induced VE-cadherin phosphorylation at tyrosines 658, 685, and 731. However, dominant negative Csk expression was unable to induce changes in the monolayer permeability. In contrast, expression of constitutively active Src decreased barrier function and promoted VE-cadherin phosphorylation on tyrosines 658 and 731, although the increase in VE-cadherin phosphorylation preceded the increase in permeability by 4–6 h. Csk knockdown induced VE-cadherin phosphorylation at sites 658 and 731 but did not induce a loss in barrier function. Co-immunoprecipitation and immunofluorescence studies suggest that phosphorylation of those sites did not impair VE-cadherin ability to bind p120 and β-catenin or the ability of these proteins to localize at the plasma membrane. Taken together, our data show that Src-induced tyrosine phosphorylation of VE-cadherin is not sufficient to promote an increase in endothelial cell monolayer permeability and suggest that signaling leading to changes in vascular permeability in response to inflammatory mediators or growth factors may require VE-cadherin tyrosine phosphorylation concurrently with other signaling pathways to promote loss of barrier function.
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Review ArticleCdc42 Regulates the Restoration of Endothelial Adherens Junctions and Permeability

Section snippets

Structure of Endothelial Adherens Junctions

Role of Adherens Junctions in the Mechanism of Vascular Inflammation

Cdc42-Mediated Restoration of Endothelial Barrier Function

Mechanisms of Cdc42-Mediated Endothelial Adherens Junctional Repair

Conclusions and Future Directions

FEBS Lett

Cell

Blood

J Biol Chem

J Biol Chem

Curr Opin Cell Biol

J Biol Chem

J Biol Chem

J Biol Chem

Cell

Cell

Curr Opin Cell Biol

Epoxycyclopentenone-containing oxidized phospholipids restore endothelial barrier function via Cdc42 and Rac

Circ Res

Dynamic interaction of PTPmu with multiple cadherins in vivo

J Cell Biol

Cdc42 regulates adherens junction stability and endothelial permeability by inducing alpha-catenin interaction with the vascular endothelial cadherin complex

Circ Res

Thrombin signalling and protease-activated receptors

Nature

Reversibility of increased microvessel permeability in response to VE-cadherin disassembly

Am J Physiol Lung Cell Mol Physiol

Myosin light chain kinase–regulated endothelial cell contraction: the relationship between isometric tension, actin polymerization, and myosin phosphorylation

J Cell Biol

Rho GTPases and the control of cell behaviour

Biochem Soc Trans

VE-cadherin–p120 interaction is required for maintenance of endothelial barrier function

Am J Physiol Lung Cell Mol Physiol

Review Article
Cdc42 Regulates the Restoration of Endothelial Adherens Junctions and Permeability