Nicolas Schlegel

Role of GTPases in control of microvascular permeability

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.

VASP is involved in cAMP-mediated Rac 1 activation in microvascular endothelial cells

Accumulating evidence points to a significant role of vasodilator-stimulated phosphoprotein (VASP) in the maintenance of endothelial barrier functions. We have recently shown that impaired barrier functions in VASP-deficient microvascular myocardial endothelial cells (MyEnd VASP(-/-)) correlated with decreased Rac 1 activity. To further test the hypothesis that VASP is involved in regulation of Rac 1 activity, we studied cAMP-dependent Rac 1 activation. Both inhibition of Rac 1 activation by NSC-23766 and inhibition of PKA by PKI completely blunted the efficacy of forskolin/rolipram (F/R)-mediated cAMP increase to stabilize barrier functions as revealed by measurements of transendothelial resistance (TER). Because these results indicate that PKA/Rac 1 activation is important for barrier stabilization, we tested this signaling pathway in VASP(-/-) cells. We found that F/R and isoproterenol reduced permeability measured as FITC-dextran flux across VASP(-/-) monolayers, but not below baseline levels of wild-type cells (WT). Moreover, cAMP-mediated Rac 1 activation was reduced to approximately 50% of WT levels, and both PKA inhibition by PKI and PKA anchoring via A kinase anchoring peptides (AKAPs) by HT31 almost completely abolished Rac 1 activation in VASP(-/-) and WT endothelium. Accordingly, HT31 significantly reduced F/R-mediated TER increase in WT cells and completely blocked the protective effect of cAMP on endothelial barrier properties. Together, our data underline the significant role of cAMP-mediated Rac 1 activation for endothelial barrier stabilization and demonstrate that both AKAP-mediated PKA anchoring and VASP are required for this process.

Lipopolysaccharide-induced endothelial barrier breakdown is cyclic adenosine monophosphate dependent in vivo and in vitro

Objectives:To determine whether cyclic adenosine monophosphate (cAMP) is critically involved in lipopolysaccharide (LPS)-induced breakdown of endothelial barrier functions in vivo and in vitro. Design:Experimental laboratory research. Setting:Research laboratory. Subjects:Wistar rats and cultured human microvascular endothelial cells. Intervention:Permeability measurements in single postcapillary venules in vivo and permeability measurements and cell biology techniques in vitro. Measurements and Results:We demonstrate that within 120 minutes LPS increased endothelial permeability in rat mesenteric postcapillary venules in vivo and caused a barrier breakdown in human dermal microvascular endothelial cells in vitro. This was associated with the formation of large intercellular gaps and fragmentation of vascular endothelial cadherin immunostaining. Furthermore, claudin 5 immunostaining at cell borders was drastically reduced after LPS treatment. Interestingly, activity of the small GTPase Rho A, which has previously been suggested to mediate the LPS-induced endothelial barrier breakdown, was not increased after 2 hours. However, activity of Rac 1, which is known to be important for maintenance of endothelial barrier functions, was significantly reduced to 64 ± 8% after 2 hours. All LPS-induced changes of endothelial cells were blocked by a forskolin-mediated or rolipram-mediated increase of cAMP. Consistently, enzyme-linked immunosorbent assay-based measurements demonstrated that LPS significantly decreased intracellular cAMP. Conclusion:In summary, our data demonstrate that LPS disrupts endothelial barrier properties by decreasing intracellular cAMP. This mechanism may involve inactivation of Rac 1 rather than activation of Rho A.

Atrial natriuretic peptide enhances microvascular albumin permeability by the caveolae-mediated transcellular pathway

Aims Cardiac atrial natriuretic peptide (ANP) participates in the maintenance of arterial blood pressure and intravascular volume homeostasis. The hypovolaemic effects of ANP result from coordinated actions in the kidney and systemic microcirculation. Hence, ANP, via its guanylyl cyclase-A (GC-A) receptor and intracellular cyclic GMP as second messenger, stimulates endothelial albumin permeability. Ultimately, this leads to a shift of plasma fluid into interstitial pools. Here we studied the role of caveolae-mediated transendothelial albumin transport in the hyperpermeability effects of ANP. Methods and results Intravital microscopy studies of the mouse cremaster microcirculation showed that ANP stimulates the extravasation of fluorescent albumin from post-capillary venules and causes arteriolar vasodilatation. The hyperpermeability effect was prevented in mice with conditional, endothelial deletion of GC-A (EC GC-A KO) or with deleted caveolin-1 (cav-1), the caveolae scaffold protein. In contrast, the vasodilating effect was preserved. Concomitantly, the acute hypovolaemic action of ANP was abolished in EC GC-A KO and Cav-1−/− mice. In cultured microvascular rat fat pad and mouse lung endothelial cells, ANP stimulated uptake and transendothelial transport of fluorescent albumin without altering endothelial electrical resistance. The stimulatory effect on albumin uptake was prevented in GC-A- or cav-1-deficient pulmonary endothelia. Finally, preparation of caveolin-enriched lipid rafts from mouse lung and western blotting showed that GC-A and cGMP-dependent protein kinase I partly co-localize with Cav-1 in caveolae microdomains. Conclusion ANP enhances transendothelial caveolae-mediated albumin transport via its GC-A receptor. This ANP-mediated cross-talk between the heart and the microcirculation is critically involved in the regulation of intravascular volume.

cAMP with other signaling cues converges on Rac1 to stabilize the endothelial barrier- a signaling pathway compromised in inflammation.

AbstractcAMP is one of the most potent signaling molecules to stabilize the endothelial barrier, both under resting conditions as well as under challenge of barrier-destabilizing mediators. The two main signaling axes downstream of cAMP are activation of protein kinase A (PKA) as well as engagement of exchange protein directly activated by cAMP (Epac) and its effector GTPase Rap1. Interestingly, both pathways activate GTP exchange factors for Rac1, such as Tiam1 and Vav2 and stabilize the endothelial barrier via Rac1-mediated enforcement of adherens junctions and strengthening of the cortical actin cytoskeleton. On the level of Rac1, cAMP signaling converges with other barrier-enhancing signaling cues induced by sphingosine-1-phosphate (S1P) and angiopoietin-1 (Ang1) rendering Rac1 as an important signaling hub. Moreover, activation of Rap1 and inhibition of RhoA also contribute to barrier stabilization, emphasizing that regulation of small GTPases is a central mechanism in this context. The relevance of cAMP/Rac1-mediated barrier protection under pathophysiologic conditions can be concluded from data showing that inflammatory mediators causing multi-organ failure in systemic inflammation or sepsis interfere with this signaling axis on the level of cAMP or Rac1. This is in line with the well-known efficacy of cAMP to abrogate the barrier breakdown in response to most barrier-compromising stimuli. New is the notion that the tight endothelial barrier under resting conditions is maintained by (1) continuous cAMP formation induced by hormones such as epinephrine or (2) by activation of Rac1 downstream of S1P that is secreted by erythrocytes and activated platelets.

Differential role of Rho GTPases in intestinal epithelial barrier regulation in vitro.

Maintenance of intestinal epithelial barrier functions is crucial to prevent systemic contamination by microbes that penetrate from the gut lumen. GTPases of the Rho‐family such as RhoA, Rac1, and Cdc42 are known to be critically involved in the regulation of intestinal epithelial barrier functions. However, it is still unclear whether inactivation or activation of these GTPases exerts barrier protection or not. We tested the effects of Rho GTPase activities on intestinal epithelial barrier functions by using the bacterial toxins cytotoxic necrotizing factor 1 (CNF‐1), toxin B, C3 transferase (C3 TF), and lethal toxin (LT) in an in vitro model of the intestinal epithelial barrier. Incubation of cell monolayers with CNF‐1 for 3 h induced exclusive activation of RhoA whereas Rac1 and Cdc42 activities were unchanged. As revealed by FITC‐dextran flux and measurements of transepithelial electrical resistance (TER) intestinal epithelial permeability was significantly increased under these conditions. Inhibition of Rho kinase via Y27632 blocked barrier destabilization of CNF‐1 after 3 h. In contrast, after 24 h of incubation with CNF‐1 only Rac1 and Cdc42 but not RhoA were activated which resulted in intestinal epithelial barrier stabilization. Toxin B to inactivate RhoA, Rac1, and Cdc42 as well as Rac1 inhibitor LT increased intestinal epithelial permeability. Similar effects were observed after inhibition of RhoA/Rho kinase signaling by C3 TF or Y27632. Taken together, these data demonstrate that both activation and inactivation of RhoA signaling increased paracellular permeability whereas activation of Rac1 and Cdc42 correlated with stabilized barrier functions. J. Cell. Physiol. 226: 1196–1203, 2011.

Phosphodiesterase-4 inhibition as a therapeutic approach to treat capillary leakage in systemic inflammation

•  A specific therapy to treat capillary leakage in systemic inflammation and sepsis is not available at present. •  Recent studies demonstrated that reduced cAMP levels in endothelial cells contribute to inflammation‐induced breakdown of the endothelial barrier. •  The present study demonstrates that systemically applied phosphodiesterase‐4 inhibitors to increase endothelial cAMP are effective to prevent and to treat capillary leakage followed by improved microcirculation in a rodent model of systemic inflammation. •  These data suggest a highly clinically relevant and applicable approach to stabilize capillary leakage in sepsis and systemic inflammation.

Impaired integrin-mediated adhesion contributes to reduced barrier properties in VASP-deficient microvascular endothelium.

Recent studies point to a significant role of vasodilator‐stimulated phosphoprotein (VASP) in the maintenance of endothelial barrier functions in vivo and in vitro. Moreover, it has been reported that VASP is required for activation of the small GTPase Rac 1. However, little is known whether VASP is involved in the regulation of cell adhesion molecules that are critical for maintenance of the endothelial barrier. Here we demonstrate that impaired barrier properties in VASP‐deficient (VASP−/−) microvascular myocardial endothelial cells (MyEnd) correlated with both impaired integrin‐mediated adhesion as revealed by laser tweezer trapping and reduced integrin‐dependent cell migration. This was paralleled by reduction of focal adhesions at the cell periphery as well as of β1‐integrin and VE‐cadherin cytoskeletal anchorage. Incubation of MyEnd VASP wt with RGD peptide to block interaction of integrins with extracellular matrix (ECM) reduced barrier properties and Rac 1 activity in wt endothelial monolayers mimicking the situation in VASP (−/−) cells under resting conditions. Moreover, cAMP‐mediated Rac 1 activation was reduced under conditions of impaired integrin‐mediated adhesion in wt cells and cAMP‐induced increase in VE‐cadherin cytoskeletal anchorage was abolished in VASP (−/−) endothelium. In summary, these data indicate that VASP is required for integrin‐mediated adhesion which stabilizes endothelial barrier properties at least in part by facilitating Rac 1 activation. J. Cell. Physiol. 220: 357–366, 2009.

Impaired cAMP and Rac 1 Signaling Contribute to TNF-α-induced Endothelial Barrier Breakdown in Microvascular Endothelium

Objective: In sepsis, tumor necrosis factor‐alpha (TNF‐α) contributes to endothelial barrier breakdown. The involvement of Rho A/rho kinase signaling has recently been challenged. Here, we tested the role of cAMP and Rac 1 signaling. Materials and Methods: For this study, we took in vivo measurements of hydraulic conductivity in postcapillary mesenteric venules of adult rats. Measurements of transendothelial electrical resistence (TER), fluorescein isothiocyanate–dextran flux, Western blotting, immunostaining, and enzyme‐linked immunosorbent assay–based measurements of cAMP levels and Rho‐GTPase activity in human microvascular endothelial cells. Results: TNF‐α disrupted endothelial barrier functions in vivo and in vitro. Under these conditions, Rho A activity was significantly increased, whereas Rac 1 activity was decreased and Cdc42 was unaltered. Moreover, cAMP levels were reduced. Rho kinase inhibition, using Y27632, did not prevent TNF‐α‐induced barrier breakdown. In contrast, preincubation with forskolin and rolipram (F/R) to increase cAMP and cytotoxic necrotizing factor 1 to activate Rac 1 and Rho A abolished TNF‐α‐induced barrier breakdown in vivo and in vitro. Moreover, inactivation of Rac 1 was blocked by F/R‐mediated increase of cAMP, whereas Rho A activation was only partially inhibited. Conclusion: Our data indicate that decrease of cAMP and Rac 1 inactivation, rather than Rho A activation, contribute to TNF‐α‐induced endothelial barrier breakdown in vivo and in vitro.

Soluble VE-cadherin is involved in endothelial barrier breakdown in systemic inflammation and sepsis

AIMS Microvascular endothelial barrier breakdown in sepsis precedes organ failure and death in patients. We tested the hypothesis that the formation of endothelium-derived soluble vascular endothelial (VE)-cadherin fragments (sVE-cadherin) is involved in inflammation-induced endothelial barrier disruption. METHODS AND RESULTS Incubation of human dermal microvascular endothelial cells (HDMEC) with tumour necrosis factor-α (TNF-α) and bacterial lipopolysaccharide (LPS) led to endothelial barrier disruption which correlated with significantly increased sVE-cadherin at a size of ∼90 kDa in cell culture supernatants. Inhibition of the VE-cadherin-cleaving disintegrin and metalloproteinase ADAM10 using GI254023X attenuated inflammation-induced formation of sVE-cadherin and endothelial barrier disruption, suggesting ADAM10-mediated shedding as a mechanism underlying sVE-cadherin release. Formation of VE-cadherin fragments at 90 and 110 kDa was observed when recombinant VE-cadherin (rVE-cadherin) was digested with recombinant ADAM10. Mass spectrometry of the VE-cadherin fragments showed that they originated from cleavage of the extracelluar domain and thereby several cleavage sites of ADAM10 were identified. Atomic force microscopy measurements demonstrated that cell culture supernatants containing sVE-cadherin and application of rVE-cadherin blocked VE-cadherin binding. Accordingly rVE-cadherin dose-dependently led to loss of endothelial barrier functions in HDMEC monolayers. Finally, in patients suffering from severe sepsis or septic shock with clinical signs of a microvascular leackage, serum levels of sVE-cadherin were significantly increased. CONCLUSION Taken together, formation of sVE-cadherin is associated and contributes to inflammation-induced breakdown of endothelial barrier functions by inhibition of VE-cadherin binding. The underlying mechanism of VE-cadherin cleavage involves ADAM10 and appears to be of clinical relevance since sVE-cadherin was augmented in patients with severe sepsis.