The Janus-faced regulation of endothelial permeability by cyclic GMP.

Editorial FocusThe Janus-faced regulation of endothelial permeability by cyclic GMPWolfgang M. KueblerWolfgang M. KueblerThe Keenan Research Centre at the Li Ka Shing Knowledge Institute of St. Michael's; Department of Surgery, University of Toronto, Ontario, Canada; Institute of Physiology, Charité-Universitaetsmedizin Berlin and German Heart Institute Berlin, GermanyPublished Online:01 Aug 2011https://doi.org/10.1152/ajplung.00192.2011This is the final version - click for previous versionMoreSectionsPDF (459 KB)Download PDF ToolsExport citationAdd to favoritesGet permissionsTrack citations ShareShare onFacebookTwitterLinkedInWeChat following its identification as the long-searched-for endothelial-derived relaxing factor and the subsequent recognition of its additional anti-inflammatory and antiaggregatory properties, nitric oxide (NO) was initially hailed as the new universal therapeutic weapon in pathologies associated with hypertension, inflammation, and/or coagulation. Hopes were particularly high for the treatment of acute lung injury (ALI), which characteristically displays all of these pathological features. Yet, although clinical trials showed initial improvement of oxygenation with inhalation of NO, these effects were not sustained and did not reduce mortality (8). These and other studies led to the realization that the role of NO in ALI and permeability-type lung edema is much more complex, in that NO, based on its site of action and interaction partners, may have both protective and detrimental effects. In the past, the beneficial effects of NO have at large been attributed to activation of its molecular target soluble guanylyl cyclase (sGC) and the formation of the second messenger 3′,5′-cyclic guanosine monophosphate (cGMP) from guanosine triphosphate (GTP), whereas barrier-disruptive effects were ascribed to the formation of peroxynitrite (ONOO−) in the presence of superoxide (O2−) or conjecturally to S-nitrosylation of junctional proteins (15). Recent evidence, however, demonstrates that cGMP signaling by itself may play an ambivalent role in vascular barrier regulation, a recognition that not only incites new scientific perspectives and challenges but may be of considerable clinical relevance given the pending introduction of sGC activators as novel therapeutic strategy in cardiopulmonary disease (18).Barrier-Protective Effects of cGMPCompelling evidence for the barrier-protective role of cGMP comes from a series of in vitro and in situ studies that set to increase endothelial cGMP levels by administration of exogenous cyclic nucleotide analogs. In these experiments, membrane-permeant cGMP analogs were shown to protect cultured pulmonary artery endothelial cells (PAECs) from oxidant-induced endothelial barrier failure (10) and attenuated the increase in lung vascular filtration coefficient (Kf) in isolated perfused lungs exposed to ischemia and reperfusion (1) or hydrostatic stress (24). The barrier-enhancing properties of cGMP were further substantiated by use of a group of recently identified sGC stimulators and activators that either amplify NO-induced cGMP formation by native (reduced) sGC, or induce cGMP generation from heme-free or oxidized sGC independent of NO, respectively (18). In line with the proposed barrier protection by cGMP, the sGC stimulator BAY 41-2272 was shown to reduce lung vascular permeability in models of overventilation (17), elevated lung vascular pressures (24), or pulmonary ischemia-reperfusion injury (2).The molecular mechanisms underlying these barrier-enhancing effects of cGMP are probably multifaceted (Fig. 1): cGMP signals predominantly through 1) activation of either of the two cGMP-regulated protein kinases, cGKI with two isozymes, cGKIα and cGKIβ, and cGKII (4); yet expression of cGKII in vascular endothelium has thus far not been detected; 2) regulation of cyclic nucleotide-gated membrane ion channels (23); or 3) allosteric regulation of cyclic nucleotide degrading phosphodiesterases (PDEs). In the latter respect, cGMP directly stimulates PDE2 and inhibits PDE3, which hydrolyzes both 3′,5′-cyclic adenosine monophosphate (cAMP) and cGMP, whereas the stimulating effect of cGMP on its own hydrolysis by PDE5 is mediated via cGKI (5).Fig. 1.Pathways mediating barrier-protective and -disruptive effects of cGMP signaling in endothelial cells. Activation of soluble (sGC) or particulate (pGC) guanylyl cyclase by NO or natriuretic peptides (NPs), respectively, stimulates the conversion of guanosine triphosphate (GTP) to 3′,5′-cyclic guanosine monophosphate (cGMP). cGMP activates cGMP-regulated protein kinase I (cGKI), which protects barrier function by stabilizing VE-cadherin at the cell membrane, attenuating Ca2+ influx via transient receptor potential (TRP) channels, promoting Ca2+ uptake into the endoplasmic reticulum (ER) via Ca2+-ATPase, and inactivating RhoA. The latter events prohibit phosphorylation and, thus, inactivation of myosin light chain (MLC) phosphatase (composed of 3 subunits, MYPT1, M20, and PP1) while limiting the Ca2+/calmodulin (CaM)-dependent activation of MLC kinase (MLCK), thus shifting the balance from phosphorylated (MLC-P) to unphosphorylated MLC, resulting in actin/myosin dissociation and barrier stabilization. Cyclic GMP furthermore stimulates its own hydrolysis to GMP by cGKI-dependent activation of phosphodiesterase 5 (PDE5) and by direct allosteric activation of phosphodiesterase 2 (PDE2) while blocking phosphodiesterase 3 (PDE3). PDE2, however, will also hydrolyze 3′,5′-cyclic adenosine monophosphate (cAMP), which is formed from adenosine triphosphate (ATP) by transmembrane adenylyl cyclase (AC). Subplasmalemmal cAMP stabilizes the endothelial barrier via both protein kinase A (PKA) and Epac (exchange protein directly activated by cAMP)-dependent signaling pathways. Consequently, stimulation of cAMP hydrolysis by PDE2 will terminate this barrier-protective effect and promote barrier dysfunction in a cGMP-dependent manner.Download figureDownload PowerPointExperiments in PAECs show that the barrier-protective effect of exogenous cGMP analogs requires cGKI (10) and is associated with an attenuated intracellular Ca2+ response (12), suggesting that cGKI may regulate lung vascular permeability by interference with endothelial Ca2+ entry and/or removal mechanisms. Attenuation of endothelial Ca2+ signaling may consequently preclude endothelial barrier dysfunction by preventing the Ca2+/calmodulin-dependent activation of endothelial myosin light chain kinase. Polymodal cation channels of the transient receptor potential canonical (TRPC) and vanilloid (TRPV) subfamilies present likely target candidates in this context. TRPC6 and TRPV4 play important roles in endothelial barrier failure in permeability-type (6) and hydrostatic lung edema (24), respectively, and are negatively regulated by cGMP in a cGKI-dependent manner (21, 24). In addition, cytosolic Ca2+ levels in endothelial cells may be reduced by cGKI-mediated stimulation of endoplasmic reticulum Ca2+-ATPase facilitating Ca2+ store uptake (7). In smooth muscle cells, cGMP signaling via cGKI has furthermore been shown to inactivate RhoA by phosphorylation at Ser188 (11) and to phosphorylate and thereby activate myosin light chain phosphatase (3), mechanisms that may stabilize the vascular barrier by a Ca2+-independent inhibition of endothelial actin-myosin contraction. cGMP also stabilizes intercellular junctional complexes, as it prevents the oxidant-induced loss of junctional VE-cadherin in PAECs (10). Notably, vasodilator-stimulated phosphoprotein, a well-known cGKI target and attractive downstream candidate mechanism for barrier protection owing to its role in actin polymerization and stress fiber bundling, does not seem to contribute significantly to the barrier-protective effects of cGMP (14).Barrier-Disruptive Effects of cGMPDespite this well-based theoretical rationale and experimental evidence for a barrier-protective role of cGMP, an increasing number of reports suggest that cGMP may also be barrier-disruptive. This notion was first derived from measurements of hydraulic conductivity (Lp) by the Landis technique in frog mesenteric capillaries demonstrating that agents which elevate intracellular cGMP levels such as atrial natriuretic peptide (an activator of particulate guanylyl cyclase), exogenous NO donors (activating sGC) or zaprinast (an inhibitor of cGMP hydrolyzing PDEs) increase baseline capillary permeability (9). Schmidt and coworkers (17) established the relevance of cGMP-dependent barrier disruption in high tidal volume (HVT)-induced lung injury by showing that sGC inhibition attenuates whereas sGC stimulation amplifies cGMP formation and lung microvascular leak in parallel. Their concomitant finding that 1) HVT increases the expression of the cAMP-hydrolyzing phosphodiesterase 2A (PDE2A) and that 2) PDE2A inhibition protects from HVT-induced endothelial dysfunction provided a first mechanistic explanation for the seemingly double-edged role of cGMP in the regulation of lung vascular permeability (Fig. 1): Subplasmalemmal cAMP as generated by transmembrane adenylyl cyclase enhances endothelial barrier properties by acting through protein kinase A (PKA) and Epac (exchange protein directly activated by cAMP) as recently highlighted in a comprehensive review (16). As PDE2A is stimulated by cGMP, elevated levels of both cGMP and PDE2A will promote the hydrolysis of cAMP, thereby terminating its barrier-stabilizing effect. Remarkably, this pathway appears to become prominent at high cGMP concentrations, whereas small increments in cGMP may conversely attenuate cAMP hydrolysis by inhibiting PDE3 (19). To complicate matters further, PDE2A activation will also hydrolyze cGMP, thereby potentially further aggravating endothelial barrier dysfunction by blocking the barrier-stabilizing effects of cGMP (20). The relevance of the cGMP-PDE2 pathway is not confined to overventilation-induced lung injury, since a similar protective role of pharmacological PDE2 inhibition was recently documented in a model of pneumococcal pneumonia (22). In a study published in this issue of the Journal (13), the group of David Pearse has now extended their previous findings on the role of PDE2A in ALI. First, in a model of LPS-induced ALI they document a time-dependent increase in PDEA2 expression that peaked at day 4 and closely matched the time course of the characteristic features of ALI. Additional overventilation further amplified PDE2A expression in conjunction with expression of inducible NO synthase. The applied double hit thus increases the level of both the enzyme and its stimulatory pathway that will result in accelerated cAMP hydrolysis. Second, in an elegant approach using adenoviral-based in vivo transfection of a short hairpin RNA, Rentsendorj and colleagues (13) accomplished a partial knockdown of PDE2A in murine lungs. This strategy proved effective to reduce ALI subsequent to LPS and overventilation compared with mice transfected with a control vector. Notably, adenoviral transfection per se aggravated critical features of ALI and caused considerable mortality that was effectively prevented by PDE2A knockdown, further substantiating the proposed relevance of PDE2A in ALI. In line with its proposed mode of action, attenuation of ALI by PDE2A knockdown was associated with increased cAMP concentrations in the lung. PDE2A knockdown also reduced the expression of inducible nitric oxide synthase (iNOS) and attenuated the downstream increase in cGMP concentration in ALI lungs by a mechanism that remains to be determined. Of relevance, this finding identifies a potentially detrimental feedforward interaction between PDE2A and iNOS in ALI, in that PDE2A is required for iNOS upregulation, which in turn (via NO-dependent activation of sGC) will lead to cGMP generation that activates PDE2A.The Janus Face of cGMP SignalingThe divergent effects of cGMP are reminiscent of the dual role of cAMP in the regulation of lung vascular permeability. Depending on its subcellular compartmentalization, cAMP not only confers barrier protection by its effects in the subplasma membrane compartment but may instead disrupt the endothelial barrier when it gains access to the cytosolic space (16). It may be speculated that the effects of cGMP are likewise closely governed by subcellular gradients that are generated by an intricate spatial arrangement of cGMP forming guanylyl cyclases, cGMP hydrolyzing phosphodiesterases, and molecular targets of cGMP such as cGKI and PDE2A. The evolving role of cGMP and PDE2A as important regulators of pulmonary barrier function opens new perspectives for therapeutic targeting of lung vascular leakage. Notably, this mechanism may have contributed to the failure of inhaled NO strategies to improve primary outcome in clinical acute respiratory distress syndrome trials. More importantly, its recognition raises similar caution with respect to ongoing clinical trials addressing the safety and efficacy of pharmacological sGC stimulators in patients at risk for lung edema (18).DISCLOSURESNo conflicts of interest are declared by the author(s).REFERENCES1. Chetham PM , Sefton WD , Bridges JP , Stevens T , McMurtry IF. Inhaled nitric oxide pretreatment but not posttreatment attenuates ischemia-reperfusion-induced pulmonary microvascular leak. Anesthesiology 86: 895–902, 1997.Crossref | PubMed | ISI | Google Scholar2. Egemnazarov B , Sydykov A , Schermuly RT , Weissmann N , Stasch JP , Sarybaev AS , Seeger W , Grimminger F , Ghofrani HA. Novel soluble guanylyl cyclase stimulator BAY 41–2272 attenuates ischemia-reperfusion-induced lung injury. Am J Physiol Lung Cell Mol Physiol 296: L462–L469, 2009.Link | ISI | Google Scholar3. Frei E , Huster M , Smital P , Schlossmann J , Hofmann F , Wegener JW. Calcium-dependent and calcium-independent inhibition of contraction by cGMP/cGKI in intestinal smooth muscle. Am J Physiol Gastrointest Liver Physiol 297: G834–G839, 2009.Link | ISI | Google Scholar4. Hofmann F , Bernhard D , Lukowski R , Weinmeister P. cGMP regulated protein kinases (cGK). Hand Exp Pharmacol 191: 137–162, 2009.Crossref | Google Scholar5. Kemp-Harper B , Schmidt HH. cGMP in the vasculature. Hand Exp Pharmacol 191: 447–467, 2009.Crossref | Google Scholar6. Kini V , Chavez A , Mehta D. A new role for PTEN in regulating transient receptor potential canonical channel 6-mediated Ca2+ entry, endothelial permeability, and angiogenesis. J Biol Chem 285: 33082–33091, 2010.Crossref | PubMed | ISI | Google Scholar7. Lau KL , Kong SK , Ko WH , Kwan HY , Huang Y , Yao X. cGMP stimulates endoplasmic reticulum Ca2+-ATPase in vascular endothelial cells. Life Sci 73: 2019–2028, 2003.Crossref | PubMed | ISI | Google Scholar8. Levitt JE , Matthay MA. Treatment of acute lung injury: historical perspective and potential future therapies. Semin Respir Crit Care Med 27: 426–437, 2006.Crossref | PubMed | ISI | Google Scholar9. Meyer DJ , Huxley VH. Capillary hydraulic conductivity is elevated by cGMP-dependent vasodilators. Circ Res 70: 382–391, 1992.Crossref | PubMed | ISI | Google Scholar10. Moldobaeva A , Welsh-Servinsky LE , Shimoda LA , Stephens RS , Verin AD , Tuder RM , Pearse DB. Role of protein kinase G in barrier-protective effects of cGMP in human pulmonary artery endothelial cells. Am J Physiol Lung Cell Mol Physiol 290: L919–L930, 2006.Link | ISI | Google Scholar11. Murthy KS , Zhou H , Grider JR , Makhlouf GM. Inhibition of sustained smooth muscle contraction by PKA and PKG preferentially mediated by phosphorylation of RhoA. Am J Physiol Gastrointest Liver Physiol 284: G1006–G1016, 2003.Link | ISI | Google Scholar12. Pearse DB , Shimoda LA , Verin AD , Bogatcheva N , Moon C , Ronnett GV , Welsh LE , Becker PM. Effect of cGMP on lung microvascular endothelial barrier dysfunction following hydrogen peroxide. Endothelium 10: 309–317, 2003.Crossref | PubMed | Google Scholar13. Rentsendorj O , Damarla M , Aggarwal NR , Choi JY , Johnston L , D'Alessio FR , Crow MT , Pearse DB. Knockdown of lung phosphodiesterase 2A attenuates alveolar inflammation and protein leak in a two-hit mouse model of acute lung injury. Am J Physiol Lung Cell Mol Physiol. First published May 13, 2011; doi:10.1152/ajplung.00073.2011.ISI | Google Scholar14. Rentsendorj O , Mirzapoiazova T , Adyshev D , Servinsky LE , Renne T , Verin AD , Pearse DB. Role of vasodilator-stimulated phosphoprotein in cGMP-mediated protection of human pulmonary artery endothelial barrier function. Am J Physiol Lung Cell Mol Physiol 294: L686–L697, 2008.Link | ISI | Google Scholar15. Sanchez FA , Rana R , Kim DD , Iwahashi T , Zheng R , Lal BK , Gordon DM , Meininger CJ , Duran WN. Internalization of eNOS and NO delivery to subcellular targets determine agonist-induced hyperpermeability. Proc Natl Acad Sci USA 106: 6849–6853, 2009.Crossref | PubMed | ISI | Google Scholar16. Sayner SL. Emerging themes of cAMP regulation of the pulmonary endothelial barrier. Am J Physiol Lung Cell Mol Physiol 300: L667–L678, 2011.Link | ISI | Google Scholar17. Schmidt EP , Damarla M , Rentsendorj O , Servinsky LE , Zhu B , Moldobaeva A , Gonzalez A , Hassoun PM , Pearse DB. Soluble guanylyl cyclase contributes to ventilator-induced lung injury in mice. Am J Physiol Lung Cell Mol Physiol 295: L1056–L1065, 2008.Link | ISI | Google Scholar18. Stasch JP , Pacher P , Evgenov OV. Soluble guanylate cyclase as an emerging therapeutic target in cardiopulmonary disease. Circulation 123: 2263–2273, 2011.Crossref | PubMed | ISI | Google Scholar19. Surapisitchat J , Jeon KI , Yan C , Beavo JA. Differential regulation of endothelial cell permeability by cGMP via phosphodiesterases 2 and 3. Circ Res 101: 811–818, 2007.Crossref | PubMed | ISI | Google Scholar20. Suttorp N , Hippenstiel S , Fuhrmann M , Krull M , Podzuweit T. Role of nitric oxide and phosphodiesterase isoenzyme II for reduction of endothelial hyperpermeability. Am J Physiol Cell Physiol 270: C778–C785, 1996.Link | ISI | Google Scholar21. Takahashi S , Lin H , Geshi N , Mori Y , Kawarabayashi Y , Takami N , Mori MX , Honda A , Inoue R. Nitric oxide-cGMP-protein kinase G pathway negatively regulates vascular transient receptor potential channel TRPC6. J Physiol 586: 4209–4223, 2008.Crossref | PubMed | ISI | Google Scholar22. Witzenrath M , Gutbier B , Schmeck B , Tenor H , Seybold J , Kuelzer R , Grentzmann G , Hatzelmann A , van LV , Tschernig T , Mitchell TJ , Schudt C , Rosseau S , Suttorp N , Schutte H. Phosphodiesterase 2 inhibition diminished acute lung injury in murine pneumococcal pneumonia. Crit Care Med 37: 584–590, 2009.Crossref | PubMed | ISI | Google Scholar23. Wu S , Moore TM , Brough GH , Whitt SR , Chinkers M , Li M , Stevens T. Cyclic nucleotide-gated channels mediate membrane depolarization following activation of store-operated calcium entry in endothelial cells. J Biol Chem 275: 18887–18896, 2000.Crossref | PubMed | ISI | Google Scholar24. Yin J , Hoffmann J , Kaestle SM , Neye N , Wang L , Baeurle J , Liedtke W , Wu S , Kuppe H , Pries AR , Kuebler WM. Negative-feedback loop attenuates hydrostatic lung edema via a cGMP-dependent regulation of transient receptor potential vanilloid 4. Circ Res 102: 966–974, 2008.Crossref | PubMed | ISI | Google ScholarAUTHOR NOTESAddress for reprint requests and other correspondence: W. M. Kuebler, The Keenan Research Centre, Li Ka Shing Knowledge Institute, St. Michael's Hospital 209 Victoria St., M5B 1W8 Toronto, Ontario, Canada (e-mail: kueblerw@smh.ca). Download PDF Previous Back to Top Next FiguresReferencesRelatedInformation Cited ByHypercapnic acidosis attenuates pressure‐dependent increase in whole‐lung filtration coefficient (K f )1 August 2017 | Pulmonary Circulation, Vol. 7, No. 3The Flow-Dependent Transcription Factor KLF2 Protects Lung Vascular Barrier Function in Acute Respiratory Distress SyndromeAmerican Journal of Respiratory and Critical Care Medicine, Vol. 195, No. 5Endothelial and Microcirculatory Function and Dysfunction in SepsisClinics in Chest Medicine, Vol. 37, No. 2The pathophysiology of pulmonary hypertension in left heart diseaseSiegfried Breitling, Krishnan Ravindran, Neil M. Goldenberg, and Wolfgang M. Kuebler1 November 2015 | American Journal of Physiology-Lung Cellular and Molecular Physiology, Vol. 309, No. 9Endothelial Cell Regulation of Pulmonary Vascular Tone, Inflammation, and Coagulation16 March 2015Phosphodiesterase 2A is a major negative regulator of iNOS expression in lipopolysaccharide-treated mouse alveolar macrophages25 July 2014 | Journal of Leukocyte Biology, Vol. 96, No. 5Novel concepts of acute lung injury and alveolar-capillary barrier dysfunctionSusanne Herold, Nieves M. Gabrielli, and István Vadász15 November 2013 | American Journal of Physiology-Lung Cellular and Molecular Physiology, Vol. 305, No. 10Angiotensin-(1–7) Protects From Experimental Acute Lung InjuryCritical Care Medicine, Vol. 41, No. 11Preconditioning with soluble guanylate cyclase activation prevents postischemic inflammation and reduces nitrate tolerance in heme oxygenase-1 knockout miceWalter Z. Wang, Allan W. Jones, Meifang Wang, William Durante, and Ronald J. Korthuis15 August 2013 | American Journal of Physiology-Heart and Circulatory Physiology, Vol. 305, No. 4Microparticles and acute lung injuryMark McVey, Arata Tabuchi, and Wolfgang M. Kuebler1 September 2012 | American Journal of Physiology-Lung Cellular and Molecular Physiology, Vol. 303, No. 5Pseudomonas aeruginosa Exotoxin Y Is a Promiscuous Cyclase That Increases Endothelial Tau Phosphorylation and PermeabilityJournal of Biological Chemistry, Vol. 287, No. 30Lung heparan sulfates modulate Kfc during increased vascular pressure: evidence for glycocalyx-mediated mechanotransductionRandal O. Dull, Mark Cluff, Joseph Kingston, Denzil Hill, Haiyan Chen, Soeren Hoehne, Daniel T. Malleske, and Rajwinederjit Kaur1 May 2012 | American Journal of Physiology-Lung Cellular and Molecular Physiology, Vol. 302, No. 9 More from this issue > Volume 301Issue 2August 2011Pages L157-L160 Copyright & PermissionsCopyright © 2011 the American Physiological Societyhttps://doi.org/10.1152/ajplung.00192.2011PubMed21685243History Received 9 June 2011 Accepted 9 June 2011 Published online 1 August 2011 Published in print 1 August 2011 Metrics