cAMP with other signaling cues converges on Rac1 to stabilize the endothelial barrier— a signaling pathway compromised in inflammation Nicolas Schlegel & Jens Waschke
Received: 29 September 2013 / Accepted: 31 October 2013 # Springer-Verlag Berlin Heidelberg 2013
Abstract cAMP 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 multiorgan 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
N. Schlegel Department of General-, Visceral, Vascular and Pediatric surgery, University Hospital Wuerzburg, Oberduerrbacherstrasse 6, 97080 Wuerzburg, Germany J. Waschke (*) Institute of Anatomy and Cell Biology, Department I, Ludwig-Maximilians-Universität Munich, Pettenkoferstrasse 11, 80336 Munich, Germany e-mail: [email protected]
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. Keywords Endothelial barrier . Adherens junctions . Tight junctions . cAMP . Rho-GTPases . Inflammation
Introduction The endothelium provides a selective barrier between blood vessels and the surrounding interstitial tissue (Mehta and Malik 2006; Michel and Curry 1999). Endothelial barrier functions are deranged under various pathological conditions such as hemorrhagic stroke, vascular malformations, atherosclerosis, tumor formation and, especially, in acute inflammation (Dejana et al. 2009). Under inflammatory conditions, breakdown of the endothelial barrier is predominately caused by opening of interendothelial paracellular junctions in postcapillary venules followed by severe subcutaneous and whole body cavity edema (Goldenberg et al. 2011; Lee and Slutsky 2010; Mehta and Malik 2006). The endothelial barrier consists in intercellular tight junctions (TJ) and adherens junctions (AJ), which are also functionally coupled to cell-matrix contacts. TJs directly limit paracellular permeability. They are composed of occludin and claudins such as claudin 5, which is the only claudin expressed in peripheral endothelium and is thought to play a key role in endothelial TJ maintenance by sealing the intercellular cleft between neighboring endothelial cells (Komarova and Malik 2010). The cytoplasmic domain of TJ proteins is associated with zonula occludens protein-1 (ZO-1), which links TJs to α-catenin, spectrin and the actin cytoskeleton. Barrier-stabilizing effects mediated by the second
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messenger cyclic adenosine monophosphate (cAMP) have at least in part been attributed to an augmented number of TJ strands in the intercellular cleft (Adamson et al. 1998). In addition, AJs contribute to barrier stabilization by providing the mechanical strength to cohesion between endothelial cells (Dejana and Orsenigo 2013). The endothelium-specific AJ adhesion molecule vascular-endothelial (VE-) cadherin undergoes homophilic binding by transinteraction of its extracellular domain in Ca2+-dependent manner. The cytoplasmatic domain of VE-cadherin has binding sites for p120 catenin as well as for β- and γ-catenin, which via α-catenin or other proteins tether the cadherin–catenin complex to the actin cytoskeleton. The association of TJs and AJs with the actin cytoskeleton explains why intracellular signaling that affects actin dynamics is critically involved in the regulation of the endothelial barrier. Mediators that strengthen the cortical actin cytoskeleton lead to endothelial barrier stabilization whereas perturbation of actin dynamics strongly alters endothelial barrier functions in vivo (Waschke et al. 2005). Therefore, it is not surprising that cAMP-mediated signaling pathways regulate the endothelial barrier by modulating actin dynamics. cAMP regulates endothelial permeability by two different signaling pathways. The first involves activation of cAMPdependent protein kinase A (PKA) and phosphorylation of PKA substrate proteins such as MLCK, ERK1/2 and RhoA. The second pathway downstream of cAMP is PKAindependent and mediated through its direct binding to exchange protein directly activated by cAMP (Epac), a guanine nucleotide exchange factor (GEF) for the small GTPase Rap1 (Parnell et al. 2012). It is important to note that the different components involved in these pathways form signaling complexes and the selective anchoring of these complexes to specific subcellular domains enables specific actions of a cAMP-mediated signaling event.
For barrier stabilization cAMP generation is confined to the membranous compartment cAMP synthesis occurs predominately at the plasma membrane (Houslay et al. 2007) upon stimulation of adenylyl cyclases (AC) via G-protein-coupled receptors (GPCR) and inhibition of phosphodiesterases (PDEs), respectively. In endothelial cells, most of the cAMP synthesis is provided by the Ca2+-inhibited type 6 adenylyl cyclase (AC6) (Cioffi et al. 2002) and most of the cAMP hydrolysis is attributable to PDE3 and PDE4 (Lugnier and Schini 1990; Netherton and Maurice 2005). Physiological agents such as prostaglandin (PG) D2, E2 (Birukova et al. 2007e, 2013c; Kobayashi et al. 2013) or atrial natriuretic peptide (ANP) (Birukova et al. 2008b) increase endothelial cAMP levels. Similarly, pharmacologic β-receptor agonists such as isoproterenol (Adamson et al. 1998; Schlegel and Waschke 2009a), adenylyl cyclase
activator forskolin and phosphodiesterase 4 inhibitors have been extensively demonstrated to exert barrier-protective effects via formation of cAMP (Adamson et al. 1998; Baumer et al. 2008, 2009; He et al. 2000; Schick et al. 2012; Schlegel et al. 2008, 2012). In this context, compartimentalization of cAMP generation is thought to be crucial. It has been shown that cAMP elevation in endothelial cells can have opposing effects and, depending on the site of cAMP formation, can either lead to barrier stabilization or breakdown (Fischmeister 2006; Sayner et al. 2006). In general, cAMP synthesis in the membranous compartment, for example, by adenylyl cyclase 6, leads to endothelial barrier stabilization. In contrast, soluble adenylyl cyclases that localize outside this membranous compartment synthesize cAMP in a cytosolic pool and have been reported to cause barrier destabilization by as yet undefined mechanisms (Sayner et al. 2006).
cAMP is one of the most potent barrier-stabilizing signaling molecules It is generally accepted that cAMP under physiologic and most pathophysiologic conditions stabilizes the endothelial barrier (Curry and Adamson 2010, 2013; Mehta and Malik 2006; Michel and Curry 1999; Rigor et al. 2012; Shen et al. 2010; Spindler et al. 2010). Increased permeability in response to histamine, bradykinin, platelet-activating factor (PAF), substance P and thrombin (Michel and Curry 1999), as well as to mediators causing multi-organ failure in sepsis such as lipopolysaccharide (LPS) and tumor necrosis factor-α (TNF-α) in vivo (Schick et al. 2012; Schlegel et al. 2009; Schlegel and Waschke 2009b), can be largely blunted by β2-adrenergic agents or phosphodiesterase 4 inhibition. Similarly, when endothelial barrier function is compromised under more complex pathophysiologic conditions, such as in experimental diabetes or after hypoxia-reoxygenation, glucagon-likepeptide-1 (GLP-1) or cAMP analogs restore endothelial barrier properties (Aslam et al. 2013; Wang et al. 2013). Although maybe not as pronounced, elevated cAMP levels in post-capillary venules in vivo are also capable of reducing baseline hydraulic conductivity as a measure for permeability within a time-course of about 20–40 min (Adamson et al. 1998, 2008; Spindler and Waschke 2011). In vitro, cAMP-mediated endothelial barrier stabilization, as revealed by augmented transendothelial resistance (TER), is usually detectable within 10–20 min (Baumer et al. 2008; Birukova et al. 2009; Spindler et al. 2010, 2011). Interestingly, selective stimulation of the Epac1/Rap1 pathway by O-Me-cAMP in vivo prevents increased permeability but, in contrast to cultured endothelium, does not reduce baseline permeability in intact microvessels (Adamson et al. 2008; Baumer et al. 2008; Cullere et al. 2005; Spindler et al. 2011). This discrepancy between cultured endothelium and endothelial cells in intact microvessels indicates
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that cultured cells display a more unstable, inflammatory phenotype (Curry and Adamson 2010, 2013). Enhanced barrier properties were paralleled by increased and linearized immunostaining for VE-cadherin and claudin 5 or occludin, which on the ultrastructural level was reflected by an increased number of TJ strands as well as augmented intercellular cleft length and formation of complex interdigitating junctions with a higher number of intercellular junctions (Adamson et al. 2008; Spindler et al. 2011). This is in line with increased VE-cadherin-mediated binding of endothelial cells after Epac1/Rap1 stimulation (Fukuhara et al. 2005; Kooistra et al. 2005).
cAMP stabilizes the endothelial barrier by activation of Rac1 Increasing evidence indicates that cAMP primarily stabilizes endothelial barrier functions via regulation of small GTPases, first of all by activation of Rac1 (Curry and Adamson 2013; Spindler et al. 2010). We showed that Rac1 is required for maintenance of endothelial barrier properties in vivo and VEcadherin-mediated binding in vitro and that Rac1 activation was effective in blunting the PAF-mediated permeability increase in intact microvessels, similar to that shown previously for cAMP (Waschke et al. 2004a, 2006). Therefore we hypothesize that Rac1 and cAMP signaling converge. Indeed, we found that cAMP was effective in blocking Rac1 glucosylation by lethal toxin (LT) and thereby the LT-induced endothelial barrier breakdown in a PKA-dependent manner in vivo (Waschke et al. 2004b). Some years later, Rac1 activation caused by increased cAMP was demonstrated in macrovascular and microvascular endothelial cells in vitro (Baumer et al. 2008; Birukova et al. 2007e). In this context, Rac1 activation can be mediated by both PKA- as well as Epac1/Rap1-induced activation of the Rac1 GEFs, Tiam1 and Vav2 (Fig. 1), which after phosphorylation are recruited to AJs (Birukova et al. 2007e, 2010; Glading et al. 2007; Lampugnani et al. 2002, 2010). At this site, Rap1 via afadin and Tiam1 directly interact with VEcadherin (Fig. 2) and VE-cadherin transinteraction in a feedback loop enhances Tiam1 and Rac1 activity (Birukova et al. 2011b, 2013b). Further, PKA-mediated activation of Rac1 requires vasodilator-stimulated phosphoprotein (VASP), β1integrin binding as well as proper PKA compartimentalization via A-kinase anchoring peptides (AKAPs) (Fig. 1) (Schlegel et al. 2008; Schlegel and Waschke 2009c; Schlegel et al. 2009), indicating that the actin-binding protein VASP regulates endothelial barrier functions on the level of Rac1 activation similar to that shown recently for cortactin (Schlegel and Waschke 2010; Schnoor et al. 2011). However, under some conditions, PKA- but not Epac-mediated signaling is engaged to stabilize barrier properties downstream of cAMP, as has been shown for the tissue inhibitor of metalloproteinase 2 (TIMP-2), which
activates adenylyl cyclases after binding to integrin α3β1 via the SH2-containing protein tyrosin phosphatase-1 (Shp-1) (Kim et al. 2012). Interestingly, when NSC-23766 was used to interfere with GEF-mediated Rac1 activation, the barrier protective effects of O-Me-cAMP to selectively activate the Epac/Rap1 pathway were completely blocked but not when overall cAMP was increased, which may indicate that Rac1 is the main downstream effector of Rap1 to induce junction remodeling and barrier stabilization, whereas other signaling molecules may exist to mediate the effects of PKA in addition to Rac1 (Spindler et al. 2011). Although in this study Cdc42 was not found to be activated in response to increased cAMP, Cdc42 may also contribute to cAMP-mediated barrier stabilization since it is effectively activated by cAMP, presumably also via Vav2 (Fig. 1) (Baumer et al. 2009; Birukova et al. 2007e, 2008b; Kobayashi et al. 2013). This body of evidence shows that cAMP stabilizes the barrier primarily via activation of Rac1, supported by Rap1 and Cdc42. Not as easy to understand is the role of RhoA downstream of cAMP. RhoA via Rho kinase and regulation of myosin light chain (MLC) phosphorylation is well known to confer a contractile phenotype to endothelial cells (Spindler et al. 2010). In cultured endothelium, this contractile phenotype can be easily induced when barrierdisrupting agents such as the coagulation factor thrombin are used. However, since the endothelium of intact post-capillary venules does not respond to thrombin unless exposed to inflammatory stimuli and inhibitors of Rho kinase or MLCK do not block the permeability increase in response to typical inflammatory mediators (which thrombin is not), these data indicate that RhoA and the contractile phenotype are less important for barrier regulation in vivo (Adamson et al. 2003; Curry et al. 2003). This fits with the observation that cAMP does not reduce RhoA activity under resting conditions, although RhoA bears a PKA consensus site and can be phosphorylated by PKA (Baumer et al. 2008; Qiao et al. 2003). Instead, under resting conditions, RhoA appears to foster cell junctions as can be concluded from studies where a deficiency of the Rho-GEF TEM4, which associates with the cadherin–catenin complex, causes endothelial barrier disruption (Fig. 1) (Ngok et al. 2013). Moreover, we believe that RhoA activation under inflammatory conditions is downstream of cAMP reduction and Rac1 inactivation (Fig. 1). When thrombin is used, cAMP levels decrease within 30 s as revealed by fluorescence resonance energy transfer (FRET) (Baumer et al. 2009). As soon as 5 min after addition of thrombin, the activities of Rac1 and Cdc42 are reduced while RhoA is activated, suggesting that all these GTPases are involved in thrombin-mediated barrier breakdown (Baumer et al. 2008, 2009). However, when LPS or TNF-α were used to increase permeability in vitro, inactivation of Rac1 coincided with the initial barrier breakdown (Schlegel et al. 2009), whereas RhoA activation was delayed. Moreover, parallel activation of Rac1 and RhoA but not
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Fig. 1 Mechanisms underlying barrier regulation by cAMP and small GTPases. Endothelial barrier properties are maintained by adherens junctions (AJ) and tight junctions (TJ). AJs consist in VE-cadherin, which is linked to the intracellular adapter molecules α-, ß-, γ- and p120-catenin and tethered to the cortical actin cytoskeleton. TJs are composed of claudin5 and occludin, which are connected to the actin cytoskeleton via Zonula occludens protein (ZO-1/2/3). cAMP-mediated effects to stabilize the endothelial barrier involve protein kinase A (PKA) and exchange protein directly activated by cAMP (EPAC), which via guanine
nucleotide exchange factors (GEFs) such as Tiam1 or Vav2 activate Rac1 and Cdc42, a process involving vasodilator-stimulated phosphoprotein (VASP) and A-kinase anchoring proteins (AKAPs). Rac1 stabilization of AJs and TJs occurs in part by strengthening of the cortical actin cytoskeleton and by inhibition of RhoA via p190RhoGAP. RhoA is also regulated by the junction-associated RhoGEF TEM4 and also by GEFH1, which is sequestered by microtubules downstream of cAMP and stathmin phosphorylation (p-stmn). cAMP stabilizes microtubules also via AKAP9
inhibition of Rho kinase was capable of blocking TNF-αinduced permeability in vivo, further indicating that RhoA signaling is not crucial to the increase of permeability under these conditions. In this context, Rac1 may regulate RhoA function on the level of GTP exchange. In endothelial cells, Rac1 inhibits RhoA via p190RhoGAP, which is bound to AJs via p120-catenin (Birukova et al. 2011a; Zebda et al. 2013). In addition, Rac1 can interfere with RhoA activation by p115RhoGEF (Birukova et al. 2007a, c). Alternatively, cAMP via PKA can inhibit RhoA function independent of Rac1 by stabilization of microtubules (Fig. 1). cAMP via stathmin phosphorylation was shown to stabilize microtubules to sequester GEF-H1 and thereby to inhibit RhoA (Tian et al. 2012). Similarly, Epac (independently of Rap1) via AKAP9 caused microtubule polymerization and endothelial barrier stabilization (Sehrawat et al. 2011). The mechanisms by which Rac1 regulates endothelial barrier functions are not the subject of this article and are discussed elsewhere (Spindler et al. 2010; review by van NieuwAmerongen in this issue). However, inhibition of myosin-ATPase did not block the permeability increase caused by LT-mediated Rac1 inactivation in vivo but, similar to MLCK, inhibition in cultured endothelium abolished intercellular gap formation and largely reduced increased permeability in these monolayers. Therefore, it can be concluded that Rac1mediated regulation of endothelial contraction (via RhoA)
in vivo is maybe less important than in cultured cells (Waschke et al. 2004c). Rather, Rac1 via p21-activated kinase and cortactin appears to regulate endothelial barrier properties on the level of the cortical actin cytoskeleton and the cadherin– catenin complex (Birukova et al. 2011b; Waschke et al. 2004c).
On the level of Rac1, cAMP signaling converges with other barrier-enhancing signaling cues Mediators that are known to be important for endothelial barrier stabilization in acute inflammation and under resting conditions affect Rac1 activity, suggesting that on this level cAMP signaling converges with other signaling cues. In this context, the angiopoietin/Tie2 system as well as sphingosine 1-phosphate (S1P) are known to be required for endothelial barrier maintenance under resting conditions (Curry and Adamson 2013; David et al. 2013). The origin of angiopoietin-1 (Ang1) are smooth muscle cells, pericytes and monocytes (Moss 2013). Ang1 binds to the endothelium-specific receptor Tie2, which activates downstream effector phosphatidylinositol 3-kinase and subsequently leads to stimulation of Rac1, a process that requires IQ Domain GTPase-activating protein1 (IQGAP1) binding to the active GTPase (Fig. 2) (David et al. 2011). This is followed by activation of p190 RhoGAP and thereby leads to inhibition of
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Fig. 2 Rac1 activity is crucial for mediators involved in endothelial barrier regulation under resting conditions and in inflammation. Endothelial barrier regulation predominately occurs on the level of postcapillary venules. Inflammatory mediators such as Lipopolysaccharide (LPS), tumor necrosis factor-α (TNF-α) or thrombin lead to the opening of intercellular junctions. Various mediators stabilize the endothelial barrier by cAMP-dependent pathways (left). Prostaglandins, atrial natriuretic peptide (ANP) or β-receptor agonists via their receptors increase cAMP levels in endothelial cells. cAMP levels in turn promote Rac1/Cdc42 activation via GEFs Tiam1 and Vav2 downstream of PKA and Epac1/Rap1. Similarly, angiopoietin1 (Ang1),
hepatocyte growth factor (HGF) and sphingosine-1-phosphate (S1P) via their receptors as well as OxPAPC cause Rac1 activation, a process involving Akt and IQGAP1. Thus, on the level of Rac1, cAMP signaling converges with these barrier-enhancing signaling cues. Endothelial barrier disruption by inflammatory mediators such as LPS, TNF-α, or thrombin via their receptors as well as loss of β-adrenergic stimulation is mediated by reduction of cAMP and Rac1 activity (right). Ang2 via blocking Tie2 receptor autophosphorylation or S1P via S1P2-receptor results in Rac1 inactivation. Interestingly, βadrenergic stimulation, S1P1 signaling and Tie2-activation are permanently required to maintain basal Rac1 activity and endothelial barrier function
RhoA (Mammoto et al. 2007). Because the Tie2 receptor is constitutively activated by Ang1, it is believed that maintenance of endothelial barrier properties is Ang1/Tie2-dependent under resting conditions (David et al. 2013; Thurston et al. 2000). Under inflammatory conditions, Ang1 was effective in preventing LPS-induced endothelial barrier breakdown in different in vivo models (David et al. 2013; Mammoto et al. 2007). The mutual antagonist of Ang1 is Ang2, which is stored within endothelial Weibel–Palade bodies and is secreted rapidly following inflammatory stimulation. Ang2 induces vascular leakage by blocking the Ang1-induced autophosphorylation of the Tie2 receptor (Moss 2013). The pathophysiological relevance of the Ang/Tie2 system is highlighted by the fact that patients with severe systemic inflammation or sepsis display reduced levels of Ang1 but increased levels of Ang2 (David et al. 2013; Fiusa et al. 2013). Another substance that stimulates the endothelium to maintain its barrier function is S1P (Garcia et al. 2001; McVerry
and Garcia 2005). It is believed that S1P is continuously released from erythrocytes or platelets and stimulates the S1 P 1 -re ce ptor that, tog eth er w ith Ak t-me diate d transactivation (Singleton et al. 2009), leads to activation of Rac1 and to endothelial barrier stabilization (Fig. 2) (Curry and Adamson 2013). The main body of evidence for this observation was derived from experiments using single microvessels that were perfused with a physiological solution lacking S1P. This condition led to increased microvascular permeability, which was abrogated after addition of S1P (Curry et al. 2012). Moreover, S1P was also found to effectively attenuate increased permeability in response to the inflammatory mediators bradykinin and PAF (Adamson et al. 2012a, b). It is not known whether S1P plasma levels are reduced in systemic inflammation or sepsis but it has been reported that the S1P carrier protein ApoM is reduced in patients under the latter conditions, which could contribute to manifestation of vascular leakage (Kumaraswamy et al. 2012).
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Hepatocyte growth factor (HGF) is also well known to induce endothelial barrier stabilization (Birukova et al. 2009; Ephstein et al. 2013; Liu et al. 2002; Singleton et al. 2007). HGF is usually found in the alveolar fluid and, because its secretion is augmented under conditions of acute inflammation such as in lung injury, pancreatitis, or after surgical trauma after partial liver resection and nephrectomy, HGF is considered as a rescue factor (Ware and Matthay 2002). Accordingly, it was demonstrated that HGF was effective in preventing VEGFinduced and thrombin-induced endothelial barrier breakdown via Rac1 (Fig. 2) (Birukova et al. 2007a, 2009). Recently, it was shown that HGF-induced Rac1 activation and endothelial barrier stabilization is mediated via S1P1 receptor and integrin β4 transactivation (Ephstein et al. 2013). It is well established that inflammation leads to augmented levels of oxidized phospholipids, which derive from an increased generation of reactive oxygen species (Fu and Birukov 2009). Among the multiple biological effects that oxidized phospholipids exert, it is remarkable that oxidized phospholipids such as oxidized 1-palmitoyl-2-arachidonoylsn-glycero-3-phosphorylcholine (OxPAPC) are capable of protecting the endothelial barrier under challenge of various inflammatory stimuli in vivo and in vitro (Birukova et al. 2013a; Fu and Birukov 2009; Nonas et al. 2008; Starosta et al. 2012; Zebda et al. 2013). Detailed investigations have revealed that endothelial barrier protection by OxPAPC involves activation of the GEFs Tiam1 and βPIX to augment Rac1/Cdc42 activity (Fig. 2) (Birukova et al. 2007d). Upstream mechanisms included activation of GPCR, PKA, Src and PKC (Birukova et al. 2007b, 2008a). Taken together, modulation of Rac1 activity represents a crucial signaling hub not only for cAMP-mediated effects but also of signaling cues that are known to critically influence endothelial barrier functions under resting conditions and in inflammation. Given that cAMP-mediated Rac1 activation and endothelial barrier stabilization is capable of interfering with alterations of Ang/Tie2- or S1P signaling, there is an enormous therapeutic potential to stabilize the endothelial barrier by pharmacological targeting this pathway, e.g., by application of phosphodiesterase inhibitors (Schick et al. 2012; Schlegel et al. 2009) or by newly developed agents such as Sac-1004 (Maharjan et al. 2013). In support of this notion, it was shown that systemic application of a phosphodiesterase-4 inhibitor was effective in preventing microvascular leakage and breakdown of the microcirculation, which drastically augmented survival rates in an animal model of systemic inflammation (Schick et al. 2012). Moreover, intraperitoneal application of phosphodiesterase-4 inhibitors or systemic application of cAMP-increasing agents such as dobutamine or norepinephrine were effective for volume expansion under resting conditions and in acute inflammation (De Backer et al. 2006; Lehtonen et al. 2004; Lin et al. 2012; Secchi et al. 2001; Stephens et al. 2011).
In sepsis and in response to inflammatory mediators, the cAMP/Rac1 signaling axis is impaired From a clinical point of view, endothelial barrier breakdown is increasingly considered to play a key role in the pathophysiology of sepsis (David et al. 2013; Goldenberg et al. 2011; Lee and Slutsky 2010; Schick et al. 2012). In systemic inflammation and sepsis, the consecutive loss of intravascular fluid after endothelial barrier disruption leads to breakdown of the microcirculation, metabolic dysbalance and organ failure, which contribute significantly to the high mortality rate of up to 60 % in these patients (Dellinger et al. 2012). Therefore, the option to enable therapeutic intervention is desperately needed. It has been demonstrated that endothelial barrier disruption by inflammatory stimuli such as bacterial LPS, TNF-α, or thrombin is at least in part caused by loss of intracellular cAMP and Rac1 inactivation (Fig. 2) (Schlegel et al. 2009). Similarly, disturbed cAMP signaling was observed in microvessels in an animal model of systemic inflammation (Schick et al. 2012). Since mRNA levels for several adenylyl cyclase isoforms including AC6 were reduced in vivo when LPS was applied, it is possible that cAMP synthesis is reduced by this mechanism (Risoe et al. 2007). Besides direct effects of inflammatory stimuli on endothelial cells, it is also known that the Ang/Tie2-signaling axis is critically deranged in systemic inflammation and sepsis (Fig. 2) (David et al. 2013). Plasma levels of the Tie2 receptor antagonist Ang2 in septic patients are strongly associated with severity of illness, markers of endothelial inflammation and adverse outcome (Fiusa et al. 2013; Kumpers et al. 2008, 2010). Furthermore, it has been shown that sepsis-induced microvascular alterations can be attributed to Ang2-mediated effects (Kumpers et al. 2011; Ziegler et al. 2013). Therefore, it can be assumed that both Ang1 mimetics as well as strategies to deplete or inhibit Ang2 may become successful therapeutic options to treat inflammation-induced endothelial barrier breakdown (David et al. 2013).
The tight endothelial barrier under resting conditions is maintained by mechanisms that continuously serve to increase cAMP or to activate Rac1 An interesting aspect is that the cAMP/Rac1 signaling axis is not only impaired under inflammatory conditions and stimulation of this signaling pathway can be used to prevent endothelial barrier breakdown but apparently cAMP/Rac1 signaling is constitutively activated, which is required for maintenance of tight barrier properties under resting conditions. For example, S1P appears to be constantly released from erythrocytes and activated platelets (Fig. 2) (Hanel et al. 2007; Pyne and Pyne 2000). Thus, when sphingosine kinase 1 that produces S1P is missing in hematopoetic cells, the lung
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endothelial barrier is compromised (Camerer et al. 2009). Similarly, when intact microvessels are perfused in the absence of erythrocytes or S1P, baseline permeability increases (Curry et al. 2012). Apparently, a similar mechanism is stabilizing the endothelial barrier via the β2-adrenergic receptor (Fig. 2) (Spindler et al. 2011). Inhibition of β2-adrenergic receptor signaling increases baseline hydraulic conductivity in vivo and reduces barrier functions in cultured endothelium. Since in these studies no epinephrine, which is the physiologic ligand of this receptor, was present, it is attractive to assume that endothelial cells can generate epinephrine. These data strongly indicate that catecholamines serve to maintain endothelial barrier properties by constant formation of cAMP. In this context, it is possible that, under resting conditions, cAMP sustains the endothelial barrier primarily via PKA-dependent mechanisms, whereas the Epac pathway is more relevant for stimulated barrier protection (Pannekoek et al. 2011).
Conclusion The main conclusion of this article is that the cAMP/Rac1 signaling axis is pivotal in endothelial barrier regulation. Dysfunction of this pathway leading to microvascular endothelial barrier breakdown significantly contributes to the pathogenicity of several severe diseases, such as multi-organ failure in sepsis. Therefore, because stimulation of cAMP and Rac1 signaling in the endothelium is effective in blocking the permeability increase in response to most inflammatory mediators, this pathway should be considered as a therapeutic target. Three aspects are interesting: (1) blood cells (primarily erythrocytes) constantly supplying mediators such as S1P and hormones may be secreted by endothelial cells to maintain endothelial barrier properties; (2) compartimentalization of signaling molecules in this pathway (AKAPs for PKA and GEFs for Rac1 and RhoA, as well as the GTPase Rap1 itself) to endothelial AJs and cytoskeletal components or via modulation of cAMP diffusion coefficients (Feinstein et al. 2012) is increasingly recognized to be important; and (3) the contractile, RhoA-dependent phenotype is less important for cAMPmediated barrier regulation but rather is induced under inflammatory conditions downstream of Rac1 inactivation.
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