Activated Protein C Mediates Novel Lung Endothelial Barrier Enhancement (2005)

Increased endothelial cell (EC) permeability is central to the pathophysiology of inflammatory syndromes such as sepsis and acute lung injury (ALI). Activated protein C (APC), a serine protease critically involved in the regulation of coagulation and inflammatory processes, improves sepsis survival through an unknown mechanism. We hypothesized a direct effect of APC to both prevent increased EC permeability and to restore vascular integrity after edemagenic agonists. We measured changes in transendothelial electrical resistance (TER) and observed that APC produced concentration-dependent attenuation of TER reductions evoked by thrombin. We next explored known EC barrier-protective signaling pathways and observed dose-dependent APC-mediated increases in cortical myosin light chain (MLC) phosphorylation in concert with cortically distributed actin polymerization, findings highly suggestive of Rac GTPase involvement. We next determined that APC directly increases Rac1 activity, with inhibition of Rac1 activity significantly attenuating APC-mediated barrier protection to thrombin challenge. Finally, as these signaling events were similar to those evoked by the potent EC barrier-enhancing agonist, sphingosine 1-phosphate (S1P), we explored potential cross-talk between endothelial protein C receptor (EPCR) and S1P1, the receptors for APC and S1P, respectively. EPCR-blocking antibody (RCR-252) significantly attenuated both APC-mediated barrier protection and increased MLC phosphorylation. We next observed rapid, EPCR and PI 3-kinase-dependent, APC-mediated phosphorylation of S1P1 on threonine residues consistent with S1P1 receptor activation. Co-immunoprecipitation studies demonstrate an interaction between EPCR and S1P1 upon APC treatment. Targeted silencing of S1P1 expression using siRNA significantly reduced APC-mediated barrier protection against thrombin. These data suggest that novel EPCR ligation and S1P1 transactivation results in EC cytoskeletal rearrangement and barrier protection, components potentially critical to the improved survival of APC-treated patients with severe sepsis. Increased endothelial cell (EC) permeability is central to the pathophysiology of inflammatory syndromes such as sepsis and acute lung injury (ALI). Activated protein C (APC), a serine protease critically involved in the regulation of coagulation and inflammatory processes, improves sepsis survival through an unknown mechanism. We hypothesized a direct effect of APC to both prevent increased EC permeability and to restore vascular integrity after edemagenic agonists. We measured changes in transendothelial electrical resistance (TER) and observed that APC produced concentration-dependent attenuation of TER reductions evoked by thrombin. We next explored known EC barrier-protective signaling pathways and observed dose-dependent APC-mediated increases in cortical myosin light chain (MLC) phosphorylation in concert with cortically distributed actin polymerization, findings highly suggestive of Rac GTPase involvement. We next determined that APC directly increases Rac1 activity, with inhibition of Rac1 activity significantly attenuating APC-mediated barrier protection to thrombin challenge. Finally, as these signaling events were similar to those evoked by the potent EC barrier-enhancing agonist, sphingosine 1-phosphate (S1P), we explored potential cross-talk between endothelial protein C receptor (EPCR) and S1P1, the receptors for APC and S1P, respectively. EPCR-blocking antibody (RCR-252) significantly attenuated both APC-mediated barrier protection and increased MLC phosphorylation. We next observed rapid, EPCR and PI 3-kinase-dependent, APC-mediated phosphorylation of S1P1 on threonine residues consistent with S1P1 receptor activation. Co-immunoprecipitation studies demonstrate an interaction between EPCR and S1P1 upon APC treatment. Targeted silencing of S1P1 expression using siRNA significantly reduced APC-mediated barrier protection against thrombin. These data suggest that novel EPCR ligation and S1P1 transactivation results in EC cytoskeletal rearrangement and barrier protection, components potentially critical to the improved survival of APC-treated patients with severe sepsis. Sepsis is a devastating inflammatory syndrome producing life-threatening end organ damage including acute lung injury (ALI) 1The abbreviations used are: ALI, acute lung injury; APC, activated protein C; ARDS, acute respiratory distress syndrome; co-IP, co-immunoprecipitation; DN, dominant-negative; EC, endothelial cell(s); EPCR, endothelial protein C receptor; HGF, hepatocyte growth factor; HPAECs, human pulmonary artery endothelial cells; MLC, myosin light chain; MLCK, myosin light chain kinase; PAR1, protease-activating receptor 1; S1P, sphingosine 1-phosphate; TER, transendothelial electrical resistance; TNF, tumor necrosis factor; PI, phosphatidylinositol; siRNA, short-interfering RNA; PDGF, platelet-derived growth factor; VEGF, vascular endothelial growth factor. (1Angus D.C. Linde-Zwirble W.T. Lidicker J. Clermont G. Carcillo J. Pinsky M.R. Crit. Care. Med. 2001; 29: 1303-1310Crossref PubMed Scopus (6868) Google Scholar). Essential and defining pathophysiological features of sepsis include the activation of the coagulation cascade and marked increases in vascular permeability, processes that result in tissue and organ dysfunction (2Vervloet M.G. Thijs L.G. Hack C.E. Semin. Thromb Hemost. 1998; 24: 33-44Crossref PubMed Scopus (268) Google Scholar, 3Gando S. Kameue T. Nanzaki S. Nakanishi Y. Thromb. Haemost. 1996; 75: 224-228Crossref PubMed Scopus (188) Google Scholar). The coagulation system, long recognized as an integral aspect of inflammation, contributes to enhanced vascular permeability via generation of procoagulant proteins, which directly perturb the integrity of the endothelial cell (EC) monolayer. For example, both thrombin, the central bioregulatory molecule of hemostasis, and fibrin, which is rapidly generated by thrombin-mediated fibrinogen cleavage, directly increase EC permeability in vivo and in vitro (4Garcia J.G. Siflinger-Birnboim A. Bizios R. Del Vecchio P.J. Fenton J.W. Malik A.B. 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Both APC and non-activated protein C bind to the endothelial protein C receptor (EPCR), a receptor structurally similar to the major histocompatibility class I/CDI family of molecules. EPCR is a crucial participant in the protein C pathway (14Fukudome K. Esmon C.T. J. Biol. Chem. 1994; 269: 26486-26491Abstract Full Text PDF PubMed Google Scholar) as EPCR ligation increases the activation of protein C by ∼20-fold (15Taylor Jr., F.B. Peer G.T. Lockhart M.S. Ferrell G. Esmon C.T. Blood. 2001; 97: 1685-1688Crossref PubMed Scopus (251) Google Scholar). The mechanism underlying the prosurvival effects of APC in sepsis is currently unknown but presumed to be related to beneficial APC effects on coagulation and inflammation. Whereas the anticoagulant properties of protein C and APC are evident in patients with inherited protein C deficiency (16Branson H.E. Katz J. Marble R. Griffin J.H. Lancet. 1983; 2: 1165-1168Abstract PubMed Scopus (282) Google Scholar, 17Griffin J.H. Evatt B. 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Investig. 2001; PubMed Scopus Google Scholar, J.G. S. Hart C.M. Patterson C.E. Am. J. Physiol. 1997; PubMed Google Scholar). TER measured using an electrical and on the TER whereas cell loss of is by a in These a that the of cell and of changes in of TER were using with between and of TER between thrombin and APC with thrombin were at barrier and as a of barrier protection thrombin, in resistance between thrombin and APC thrombin. MLC in EC were for MLC phosphorylation by by with antibody for MLC as S.M. P. J.G. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, I. A. F. J.G. Am. J. Physiol. Cell Physiol. 2001; PubMed Google Scholar). were and using EC on were exposed to with in at for and with for with in for were exposed to for were for in the by with were using a as we have S.M. P. J.G. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). Rac1 GTPase of Rac1 activation as we using a J.G. F. A. W.T. English D. J. Clin. 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Full Text Full Text PDF PubMed Scopus Google Scholar). and of siRNA against the S1P1 The siRNA human Rac1 generated using from were a siRNA for S1P1, were to the human data in a to with to human were to the human data in a to to known human For were S1P1 receptor S1P1 receptor and were and were by the For of the siRNA, a from used siRNA EC were with siRNA using as to the by were for by with of siRNA siRNA for in with for were of of the S1P1 were for by for with antibody for by APC thrombin with for by APC were in of protease The were with antibody by in and with the were with antibody by with of using enhanced of between EPCR and S1P1 were for by with antibody for by APC were in complex of protease The were with antibody by in and with the were with antibody antibody by with of using enhanced of with a used to the of data from as in the are as of APC on in the effect of APC on human lung EC barrier of TER, a of barrier integrity J.G. F. A. W.T. English D. J. Clin. Investig. 2001; PubMed Scopus Google Scholar, J.G. V. P.J. Am. J. Cell Biol. 1997; PubMed Scopus Google Scholar). that EC with APC thrombin-mediated TER decreases in a concentration-dependent of electrical resistance were at barrier of resistance at after thrombin in APC effect on the of barrier of human lung EC with APC for of significantly reduced the EC effect observed after of APC EC with thrombin-mediated barrier dysfunction and with APC of the in TER of APC on EC and MLC of MLC results in increased and a central in the properties of such as S1P, hepatocyte growth factor as well as edemagenic such as and thrombin J.G. F. A. W.T. English D. J. Clin. Investig. 2001; PubMed Scopus Google Scholar, I. A. F. J.G. Am. J. Physiol. Cell Physiol. 2001; PubMed Google Scholar, R. 1998; PubMed Google Scholar, in Scholar, J.G. Patterson C.E. J. Cell. Physiol. PubMed Scopus Google Scholar, F. K. A. N. J.G. 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EC such as thrombin the of actin in increased and whereas in contrast, APC rearrangement at the cell and reduced central Finally, APC after thrombin results in marked attenuation of and cortical rapidly increases EC cytoskeletal rearrangement and MLC phosphorylation in a of in the cytoskeletal actin and The and cell for thrombin APC APC after thrombin of Rac GTPase in EC and have that the barrier-protective cortical actin by S1P, and HGF, is by activation of the family Rac1 J.G. F. A. W.T. English D. J. Clin. Investig. 2001; PubMed Scopus Google Scholar, S.M. J.G. Am. J. Cell Biol. PubMed Scopus Google Scholar, F. K. A. N. J.G. J. 2002; PubMed Scopus Google Scholar). We next the of Rac1 in APC-mediated EC barrier that Rac activation in EC Activated Rac levels to the the of Rac1 in APC-mediated barrier we EC with a Rac1 and determined that reductions in Rac activity the APC-mediated barrier-protective of EPCR on APC-mediated and MLC EPCR is the recognized receptor for both protein C and APC, we next to the of EPCR in APC-mediated cytoskeletal activation and barrier with an EPCR antibody APC binding C. A. K. C.J. Blood. 2003; PubMed Scopus Google Scholar, C. A. C.J. 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Cell. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar) and are highly consistent with S1P1 studies were that demonstrate interaction between EPCR and S1P1, which by EPCR antibody that phosphorylation of the S1P1 receptor via the PI we to the of pathway in APC-mediated S1P1 phosphorylation. of the PI phosphorylation of S1P1 in APC signaling of PI and a known of PI with a of S1P1 transactivation by EPCR, silencing for S1P1 attenuated EPCR and EC barrier protection APC is a potent barrier-protective that through S1P1 to EC barrier protection and In we demonstrate a direct effect of APC on human pulmonary EC barrier integrity in protection from EC vascular in a and cortically distributed APC increased levels of and actin barrier produced by thrombin. APC-mediated barrier protection and a effect for to that of APC are in the these results a potential for cytoskeletal as a in the barrier protection, an effect we have with S1P, potent barrier J.G. F. A. W.T. English D. J. Clin. 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A. W.T. English D. J. Clin. Investig. 2001; PubMed Scopus Google Scholar). The mechanism through which Rac1 activation results in cortical actin and EC permeability is data the of Rac1 in EC barrier findings of S1P1 transactivation and Rac1 activation by APC are consistent with data S1P1 phosphorylation of S1P1 at and transactivation of S1P1 is for Rac1 activation and cortical actin S. J.H. N. S.S. M. M. T. Cell. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). The levels of are the enhanced cortical a increased with enhanced between and components and the cortical S.M. J.G. J. Physiol. 2001; 91: PubMed Scopus Google Scholar). of actin and MLC evoked by APC is a with barrier-protective such as S1P, HGF, and as well as J.G. F. A. W.T. English D. J. Clin. Investig. 2001; PubMed Scopus Google Scholar, S.M. J.G. Am. J. Cell Biol. PubMed Scopus Google Scholar, F. K. A. N. J.G. J. 2002; PubMed Scopus Google Scholar, A.A. K. A. A. N. J.G. PubMed Scopus Google Scholar, A.A. S.M. 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Full Text Full Text PDF PubMed Scopus Google Scholar) barrier protection and cortical actin as well as the edemagenic effects of thrombin. S1P1 phosphorylation on threonine residues as the of S1P1 activation S. J.H. N. S.S. M. M. T. Cell. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar), we S1P1 transactivation by EPCR as S1P1 which by EPCR Co-immunoprecipitation studies suggest an interaction between EPCR and S1P1 of the PI S1P1 that APC S1P1 via the PI pathway Finally, and of critical S1P1 silencing the APC-mediated EC barrier protection that S1P1 expression is for APC-mediated barrier S1P1 transactivation is a novel and been in the of ligation of growth factor receptors such as and increases phosphorylation of the and phosphorylation of and EC is T. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google Scholar). the of to in mice is after S1P1 and S1P1 transactivation M. A. S. S. J. 2001; PubMed Scopus Google Scholar). Similarly, of vascular results in phosphorylation of and growth factor which is T. PubMed Scopus Google Scholar). The mechanism by which growth factor transactivation is unknown but been to increases in sphingosine activity and newly generated allowing S1P1 ligation to S. S. 2001; 29: PubMed Google Scholar). data that S1P1 is a for barrier protection and that S1P1 in be central to signaling which to vascular integrity In increased endothelial permeability is central to the pathophysiology of inflammatory syndromes such as sepsis and data that APC increases in endothelial permeability, and vascular integrity after edemagenic such as thrombin. signaling pathways to known EC barrier-protective in the EC in cortically distributed MLC increased and These APC-mediated and effects were to increased Rac1 highly novel from the that the of APC-mediated Rac1 and barrier protection were upon signaling events evoked by the potent EC agonist, S1P, with of cross-talk between EPCR and these data suggest that EPCR cross-talk to the endothelial and of EC barrier be critical components in the improved survival observed in patients with sepsis with We and for as well as G. and E. for is to Esmon and for