Activated Neutrophils Induce Hyperpermeability and Phosphorylation of Adherens Junction Proteins in Coronary Venular Endothelial Cells

Abstract

The endothelial adherens junction is formed by complexes of transmembrane adhesive proteins, of which β-catenin is known to connect the junctional protein vascular endothelial (VE)-cadherin to the cytoskeleton and to play a signaling role in the regulation of junction-cytoskeleton interaction. In this study, we investigated the effect of neutrophil activation on endothelial monolayer integrity and on β-catenin and VE-cadherin modification. Treatment of cultured bovine coronary endothelial monolayers with C5a-activated neutrophils resulted in an increase in permeability as measured by albumin clearance across the monolayer. Furthermore, large scale intercellular gap formation was observed in coincidence with the hyperpermeability response. Immunofluorescence analysis showed that β-catenin and VE-cadherin staining changed from a uniform distribution along the membrane of control cells to a diffuse pattern for both proteins and finger-like projections for β-catenin in neutrophil-exposed monolayers. Correlatively, there was an increase in actin stress fiber formation in treated cells. Finally, β-catenin and VE-cadherin from neutrophil-treated endothelial cells showed a significant increase in tyrosine phosphorylation. Our results are the first to link neutrophil-mediated changes in adherens junctions with intercellular gap formation and hyperpermeability in microvascular endothelial cells. These data suggest that neutrophils may regulate endothelial barrier function through a process conferring conformational changes to β-catenin and VE-cadherin. The endothelial adherens junction is formed by complexes of transmembrane adhesive proteins, of which β-catenin is known to connect the junctional protein vascular endothelial (VE)-cadherin to the cytoskeleton and to play a signaling role in the regulation of junction-cytoskeleton interaction. In this study, we investigated the effect of neutrophil activation on endothelial monolayer integrity and on β-catenin and VE-cadherin modification. Treatment of cultured bovine coronary endothelial monolayers with C5a-activated neutrophils resulted in an increase in permeability as measured by albumin clearance across the monolayer. Furthermore, large scale intercellular gap formation was observed in coincidence with the hyperpermeability response. Immunofluorescence analysis showed that β-catenin and VE-cadherin staining changed from a uniform distribution along the membrane of control cells to a diffuse pattern for both proteins and finger-like projections for β-catenin in neutrophil-exposed monolayers. Correlatively, there was an increase in actin stress fiber formation in treated cells. Finally, β-catenin and VE-cadherin from neutrophil-treated endothelial cells showed a significant increase in tyrosine phosphorylation. Our results are the first to link neutrophil-mediated changes in adherens junctions with intercellular gap formation and hyperpermeability in microvascular endothelial cells. These data suggest that neutrophils may regulate endothelial barrier function through a process conferring conformational changes to β-catenin and VE-cadherin. adherens junction vascular endothelial polymorphonuclear leukocyte phenylarsine oxide coronary venular endothelial cell complement 5a damnacanthal bisindolylmaleimide I immunoprecipitation permeability coefficient of albumin The wall of exchange vessels consists of a layer of endothelial cells that connect to each other with closely opposed intercellular junctions. A major function of the junctional connection is to maintain the semi-permeable property of the endothelial barrier and to control the transvascular passage of solutes, fluid, and blood cells. Four types of junctions associated with endothelial cells have been identified: adherens junctions (AJ),1 tight junctions, gap junctions, and complexus adherentes (1Rubin L.L. Cell Biol. 1992; 4: 830-833Google Scholar, 2Dejana E. Corada M. Lampugnani M.G. FASEB J. 1995; 9: 910-918Crossref PubMed Scopus (392) Google Scholar). AJ, formed by transmembrane adhesive proteins called cadherins, appear to be the main complex regulating macromolecular permeability in microvascular endothelium. Cadherins, specifically vascular endothelial (VE)-cadherin, are associated with the actin cytoskeleton through a family of proteins called catenins, including α-catenin, β-catenin, and plakoglobin (3Kemler R. Trends Genet. 1993; 9: 317-321Abstract Full Text PDF PubMed Scopus (877) Google Scholar, 4Lampugnani M.G. Corada M. Caveda L. Breviario F. Ayalon O. Geiger B. Dejana E. J. Cell Biol. 1995; 129: 203-217Crossref PubMed Scopus (491) Google Scholar).The endothelial permeability is affected by many agonists including α-thrombin, histamine, and phorbol esters (5Ehringer W.D. Edwards M.J. Miller F.N. J. Cell. Physiol. 1996; 167: 562-569Crossref PubMed Scopus (80) Google Scholar, 6Drake W.T. Lopes N.N. Fenton J.W., II Issekutz A.C. Lab. Invest. 1992; 67: 617-627PubMed Google Scholar, 7Schaeffer R.C. Gong F. Bitrick M.S. Smith T.L. Am. J. Physiol. 1993; 264: H1798-H1809PubMed Google Scholar, 8Yuan Y. Granger H.J. Zawieja D.C. DeFily D.V. Chilian W.M. Am. J. Physiol. 1993; 264: H1734-H1739PubMed Google Scholar, 9Huang Q. Yuan Y. Am. J. Physiol. 1997; 273: H2442-H2451PubMed Google Scholar) as well as by a group of inflammatory cells, namely polymorphonuclear leukocytes (PMNs) (10Yuan Y. Mier R.A. Chilian W.M. Zawieja D.C. Granger H.J. Am. J. Physiol. 1995; 268: H490-H498Crossref PubMed Google Scholar, 11Beynon H.L.C. Davies K.A. Haskard D.O. Walport M.J. J. Immunol. 1994; 153: 3160-3167PubMed Google Scholar, 12Gibbs L.S. Lai L. Malik A.B. J. Cell. Physiol. 1990; 145: 496-500Crossref PubMed Scopus (55) Google Scholar, 13Rosengren S. Olofsson A.M. von Andrian U.H. Lundgren-Akerlund E. Arfors K.E. J. Appl. Physiol. 1991; 71: 1322-1330Crossref PubMed Scopus (52) Google Scholar, 14Carden D. Xiao F. Moak C. Willis B.H. Robinson-Jackson S. Alexander S. Am. J. Physiol. 1998; 275: H385-H392PubMed Google Scholar). At the site of injury or inflammation, circulating PMNs often adhere to and subsequently migrate through the endothelium and enter surrounding tissues (15Burns A.R. Walker D.C. Brown E.S. Thurmon L.T. Bowden R.A. Keese C.R. Simon S.I. Entman M.L. Smith C.W. J. Immunol. 1997; 159: 2893-2903PubMed Google Scholar). It has long been documented that the process of PMN adherence and migration is associated with an increase in endothelial permeability (10Yuan Y. Mier R.A. Chilian W.M. Zawieja D.C. Granger H.J. Am. J. Physiol. 1995; 268: H490-H498Crossref PubMed Google Scholar, 11Beynon H.L.C. Davies K.A. Haskard D.O. Walport M.J. J. Immunol. 1994; 153: 3160-3167PubMed Google Scholar). Although much work has been dedicated to identify PMN-derived hyperpermeability factors (11Beynon H.L.C. Davies K.A. Haskard D.O. Walport M.J. J. Immunol. 1994; 153: 3160-3167PubMed Google Scholar, 12Gibbs L.S. Lai L. Malik A.B. J. Cell. Physiol. 1990; 145: 496-500Crossref PubMed Scopus (55) Google Scholar, 13Rosengren S. Olofsson A.M. von Andrian U.H. Lundgren-Akerlund E. Arfors K.E. J. Appl. Physiol. 1991; 71: 1322-1330Crossref PubMed Scopus (52) Google Scholar, 14Carden D. Xiao F. Moak C. Willis B.H. Robinson-Jackson S. Alexander S. Am. J. Physiol. 1998; 275: H385-H392PubMed Google Scholar), little is known about the molecular targets of activated PMNs and the derived factors. Within this context, whether PMNs affect microvascular barrier function by altering the structural and functional integrity of the endothelium remains elusive.Recent evidence suggests that the AJ is involved in PMN-promoted endothelial barrier dysfunction. Upon PMN adhesion, VE-cadherin proteolysis is accompanied by the disappearance of both VE-cadherin and catenins from AJ (14Carden D. Xiao F. Moak C. Willis B.H. Robinson-Jackson S. Alexander S. Am. J. Physiol. 1998; 275: H385-H392PubMed Google Scholar, 16Del Maschio A. Zanetti A. Corada M. Rival Y. Ruco L. Lampugnani M.G. Dejana E. J. Cell Biol. 1996; 135: 497-510Crossref PubMed Scopus (280) Google Scholar, 17Allport J.R. Ding H. Collins T. Gerritsen M.E. Luscinskas F.W. J. Exp. Med. 1997; 186: 517-527Crossref PubMed Scopus (153) Google Scholar). Another hyperpermeability agonist, vascular endothelial growth factor, has been shown to increase the transendothelial flux of albumin concomitant with a loss of VE-cadherin (18Kevil C.G. Payne D.K. Mires E. Alexander J.S. J. Biol. Chem. 1998; 273: 15099-15103Abstract Full Text Full Text PDF PubMed Scopus (267) Google Scholar). These studies have provided a possible linkage between PMN activation and VE-cadherin disorganization in the regulation of endothelial barrier function. Furthermore, evidence is accumulating that the VE-cadherin-mediated cell-cell adhesion is controlled by a dynamic balance between phosphorylation and dephosphorylation of the junctional proteins including cadherins and catenins. Increased tyrosine phosphorylation of β-catenin resulted in a dissociation of the catenin from cadherin and from the cytoskeleton, leading to a weak AJ (19Kypta R.M. Su H. Reichardt L.F. J. Cell Biol. 1996; 134: 1519-1529Crossref PubMed Scopus (234) Google Scholar, 20Matsuyoshi N. Hamaguchi M. Taniguchi S. Nagfuchi A. Tsukita S. Takeichi M. J. Cell Biol. 1992; 118: 703-714Crossref PubMed Scopus (450) Google Scholar). Similarly, tyrosine phosphorylation of VE-cadherin and β-catenin occurred in loose AJ and was notably reduced in tightly confluent monolayers (21Lampugnani M.G. Corada M. Andriopoulou P. Esser S. Risau W. Dejana E. J. Cell Sci. 1997; 110: PubMed Google on the effect of activated PMNs on both endothelial permeability and phosphorylation of AJ The results showed that tyrosine phosphorylation of VE-cadherin and β-catenin was cultured coronary venular endothelial cells to activated and VE-cadherin staining a in the of proteins the cell by monolayers showed a significant increase in permeability as measured by albumin work is the first to hyperpermeability and tyrosine phosphorylation of junctional proteins in to PMNs in microvascular endothelial vascular has long been in the of coronary microvascular injury and other types of L.S. Lai L. Malik A.B. J. Cell. Physiol. 1990; 145: 496-500Crossref PubMed Scopus (55) Google Scholar, 13Rosengren S. Olofsson A.M. von Andrian U.H. Lundgren-Akerlund E. Arfors K.E. J. Appl. Physiol. 1991; 71: 1322-1330Crossref PubMed Scopus (52) Google Scholar, J. Cell Biol. 1993; PubMed Scopus Google Scholar, J.S. A. J. Cell Sci. PubMed Google Scholar, J. Cell Sci. PubMed Google Scholar, D.C. J. Invest. 67: PubMed Scopus Google Scholar). Our is the first of the of PMN activation on the function and of coronary microvascular endothelial cells. the of PMNs with the endothelium coronary and (10Yuan Y. Mier R.A. Chilian W.M. Zawieja D.C. Granger H.J. Am. J. Physiol. 1995; 268: H490-H498Crossref PubMed Google Scholar). monolayer L.S. Lai L. Malik A.B. J. Cell. Physiol. 1990; 145: 496-500Crossref PubMed Scopus (55) Google Scholar, 13Rosengren S. Olofsson A.M. von Andrian U.H. Lundgren-Akerlund E. Arfors K.E. J. Appl. Physiol. 1991; 71: 1322-1330Crossref PubMed Scopus (52) Google Scholar, C.G. Payne D.K. Mires E. Alexander J.S. J. Biol. Chem. 1998; 273: 15099-15103Abstract Full Text Full Text PDF PubMed Scopus (267) Google Scholar) to to the molecular in cells. with the coronary the monolayer a permeability a to the hyperpermeability of the molecular that to microvascular is large and blood cells to through the endothelial endothelial cells to In this the intercellular junction may conformational the gap formation and macromolecular a transmembrane and β-catenin, a signaling protein that cadherins to the actin cytoskeleton, to in tyrosine phosphorylation concomitant with an increase in suggest that this phosphorylation is an for the AJ and the of cells to Our results and of showed the disappearance of AJ proteins the cell of hyperpermeability (14Carden D. Xiao F. Moak C. Willis B.H. Robinson-Jackson S. Alexander S. Am. J. Physiol. 1998; 275: H385-H392PubMed Google Scholar, 16Del Maschio A. Zanetti A. Corada M. Rival Y. Ruco L. Lampugnani M.G. Dejana E. J. Cell Biol. 1996; 135: 497-510Crossref PubMed Scopus (280) Google Scholar, 17Allport J.R. Ding H. Collins T. Gerritsen M.E. Luscinskas F.W. J. Exp. Med. 1997; 186: 517-527Crossref PubMed Scopus (153) Google Scholar). we observed an increase in the formation of actin stress that with the AJ and permeability In cells stress fiber there is an increase in stress fiber formation across the monolayer control cell monolayers. stress with of the cell membrane and the cell a the between cells. The to protein or protein complex are the actin hyperpermeability Our is that AJ proteins the actin and for the integrity of the monolayer to In to actin stress and with including the AJ to changes in leading to a weak or of VE-cadherin and β-catenin may be a signaling that structural In of data that phosphorylation of VE-cadherin and β-catenin the hyperpermeability in the endothelial by which PMNs increase microvascular permeability has been A of evidence that the is to PMN-derived including and R. T. T. J. Immunol. 1995; PubMed Scopus Google Scholar, M.G. R.A. C. J. Invest. PubMed Scopus Google Scholar, N. A. A. D. J. Exp. 1993; Google Scholar, S. J. Invest. PubMed Scopus Google Scholar). At the site of PMN adhesion, large of the factors are and a of signaling in the endothelium. endothelial cell and intercellular are on and that protein phosphorylation is of the signaling that is activated in to PMN adhesion, and in the hyperpermeability response. In of we that activated PMNs in tyrosine phosphorylation of AJ proteins accompanied by an increase in we that the permeability increase by and a to phosphorylation of β-catenin and VE-cadherin It is that activated PMNs that or both the tyrosine and phosphorylation which may with each Our is that both types of phosphorylation are to hyperpermeability of is the first to PMN activation with phosphorylation of AJ proteins and hyperpermeability in cultured microvascular endothelial monolayers. that tyrosine phosphorylation of the adhesion proteins and adhesion in monolayers in with a of endothelial barrier function Y. Q. J. Am. J. Physiol. 1998; 275: Google Scholar). we have evidence that in both the adhesion complex and the AJ in hyperpermeability of the microvascular endothelium and that activated PMNs of have the for studies the molecular associated with changes in The wall of exchange vessels consists of a layer of endothelial cells that connect to each other with closely opposed intercellular junctions. A major function of the junctional connection is to maintain the semi-permeable property of the endothelial barrier and to control the transvascular passage of solutes, fluid, and blood cells. Four types of junctions associated with endothelial cells have been identified: adherens junctions (AJ),1 tight junctions, gap junctions, and complexus adherentes (1Rubin L.L. Cell Biol. 1992; 4: 830-833Google Scholar, 2Dejana E. Corada M. Lampugnani M.G. FASEB J. 1995; 9: 910-918Crossref PubMed Scopus (392) Google Scholar). AJ, formed by transmembrane adhesive proteins called cadherins, appear to be the main complex regulating macromolecular permeability in microvascular endothelium. Cadherins, specifically vascular endothelial (VE)-cadherin, are associated with the actin cytoskeleton through a family of proteins called catenins, including α-catenin, β-catenin, and plakoglobin (3Kemler R. Trends Genet. 1993; 9: 317-321Abstract Full Text PDF PubMed Scopus (877) Google Scholar, 4Lampugnani M.G. Corada M. Caveda L. Breviario F. Ayalon O. Geiger B. Dejana E. J. Cell Biol. 1995; 129: 203-217Crossref PubMed Scopus (491) Google Scholar). The endothelial permeability is affected by many agonists including α-thrombin, histamine, and phorbol esters (5Ehringer W.D. Edwards M.J. Miller F.N. J. Cell. Physiol. 1996; 167: 562-569Crossref PubMed Scopus (80) Google Scholar, 6Drake W.T. Lopes N.N. Fenton J.W., II Issekutz A.C. Lab. Invest. 1992; 67: 617-627PubMed Google Scholar, 7Schaeffer R.C. Gong F. Bitrick M.S. Smith T.L. Am. J. Physiol. 1993; 264: H1798-H1809PubMed Google Scholar, 8Yuan Y. Granger H.J. Zawieja D.C. DeFily D.V. Chilian W.M. Am. J. Physiol. 1993; 264: H1734-H1739PubMed Google Scholar, 9Huang Q. Yuan Y. Am. J. Physiol. 1997; 273: H2442-H2451PubMed Google Scholar) as well as by a group of inflammatory cells, namely polymorphonuclear leukocytes (PMNs) (10Yuan Y. Mier R.A. Chilian W.M. Zawieja D.C. Granger H.J. Am. J. Physiol. 1995; 268: H490-H498Crossref PubMed Google Scholar, 11Beynon H.L.C. Davies K.A. Haskard D.O. Walport M.J. J. Immunol. 1994; 153: 3160-3167PubMed Google Scholar, 12Gibbs L.S. Lai L. Malik A.B. J. Cell. Physiol. 1990; 145: 496-500Crossref PubMed Scopus (55) Google Scholar, 13Rosengren S. Olofsson A.M. von Andrian U.H. Lundgren-Akerlund E. Arfors K.E. J. Appl. Physiol. 1991; 71: 1322-1330Crossref PubMed Scopus (52) Google Scholar, 14Carden D. Xiao F. Moak C. Willis B.H. Robinson-Jackson S. Alexander S. Am. J. Physiol. 1998; 275: H385-H392PubMed Google Scholar). At the site of injury or inflammation, circulating PMNs often adhere to and subsequently migrate through the endothelium and enter surrounding tissues (15Burns A.R. Walker D.C. Brown E.S. Thurmon L.T. Bowden R.A. Keese C.R. Simon S.I. Entman M.L. Smith C.W. J. Immunol. 1997; 159: 2893-2903PubMed Google Scholar). It has long been documented that the process of PMN adherence and migration is associated with an increase in endothelial permeability (10Yuan Y. Mier R.A. Chilian W.M. Zawieja D.C. Granger H.J. Am. J. Physiol. 1995; 268: H490-H498Crossref PubMed Google Scholar, 11Beynon H.L.C. Davies K.A. Haskard D.O. Walport M.J. J. Immunol. 1994; 153: 3160-3167PubMed Google Scholar). Although much work has been dedicated to identify PMN-derived hyperpermeability factors (11Beynon H.L.C. Davies K.A. Haskard D.O. Walport M.J. J. Immunol. 1994; 153: 3160-3167PubMed Google Scholar, 12Gibbs L.S. Lai L. Malik A.B. J. Cell. Physiol. 1990; 145: 496-500Crossref PubMed Scopus (55) Google Scholar, 13Rosengren S. Olofsson A.M. von Andrian U.H. Lundgren-Akerlund E. Arfors K.E. J. Appl. Physiol. 1991; 71: 1322-1330Crossref PubMed Scopus (52) Google Scholar, 14Carden D. Xiao F. Moak C. Willis B.H. Robinson-Jackson S. Alexander S. Am. J. Physiol. 1998; 275: H385-H392PubMed Google Scholar), little is known about the molecular targets of activated PMNs and the derived factors. Within this context, whether PMNs affect microvascular barrier function by altering the structural and functional integrity of the endothelium remains evidence suggests that the AJ is involved in PMN-promoted endothelial barrier dysfunction. Upon PMN adhesion, VE-cadherin proteolysis is accompanied by the disappearance of both VE-cadherin and catenins from AJ (14Carden D. Xiao F. Moak C. Willis B.H. Robinson-Jackson S. Alexander S. Am. J. Physiol. 1998; 275: H385-H392PubMed Google Scholar, 16Del Maschio A. Zanetti A. Corada M. Rival Y. Ruco L. Lampugnani M.G. Dejana E. J. Cell Biol. 1996; 135: 497-510Crossref PubMed Scopus (280) Google Scholar, 17Allport J.R. Ding H. Collins T. Gerritsen M.E. Luscinskas F.W. J. Exp. Med. 1997; 186: 517-527Crossref PubMed Scopus (153) Google Scholar). Another hyperpermeability agonist, vascular endothelial growth factor, has been shown to increase the transendothelial flux of albumin concomitant with a loss of VE-cadherin (18Kevil C.G. Payne D.K. Mires E. Alexander J.S. J. Biol. Chem. 1998; 273: 15099-15103Abstract Full Text Full Text PDF PubMed Scopus (267) Google Scholar). These studies have provided a possible linkage between PMN activation and VE-cadherin disorganization in the regulation of endothelial barrier function. Furthermore, evidence is accumulating that the VE-cadherin-mediated cell-cell adhesion is controlled by a dynamic balance between phosphorylation and dephosphorylation of the junctional proteins including cadherins and catenins. Increased tyrosine phosphorylation of β-catenin resulted in a dissociation of the catenin from cadherin and from the cytoskeleton, leading to a weak AJ (19Kypta R.M. Su H. Reichardt L.F. J. Cell Biol. 1996; 134: 1519-1529Crossref PubMed Scopus (234) Google Scholar, 20Matsuyoshi N. Hamaguchi M. Taniguchi S. Nagfuchi A. Tsukita S. Takeichi M. J. Cell Biol. 1992; 118: 703-714Crossref PubMed Scopus (450) Google Scholar). Similarly, tyrosine phosphorylation of VE-cadherin and β-catenin occurred in loose AJ and was notably reduced in tightly confluent monolayers (21Lampugnani M.G. Corada M. Andriopoulou P. Esser S. Risau W. Dejana E. J. Cell Sci. 1997; 110: PubMed Google Scholar). on the effect of activated PMNs on both endothelial permeability and phosphorylation of AJ The results showed that tyrosine phosphorylation of VE-cadherin and β-catenin was cultured coronary venular endothelial cells to activated and VE-cadherin staining a in the of proteins the cell by monolayers showed a significant increase in permeability as measured by albumin work is the first to hyperpermeability and tyrosine phosphorylation of junctional proteins in to PMNs in microvascular endothelial cells. vascular has long been in the of coronary microvascular injury and other types of L.S. Lai L. Malik A.B. J. Cell. Physiol. 1990; 145: 496-500Crossref PubMed Scopus (55) Google Scholar, 13Rosengren S. Olofsson A.M. von Andrian U.H. Lundgren-Akerlund E. Arfors K.E. J. Appl. Physiol. 1991; 71: 1322-1330Crossref PubMed Scopus (52) Google Scholar, J. Cell Biol. 1993; PubMed Scopus Google Scholar, J.S. A. J. Cell Sci. PubMed Google Scholar, J. Cell Sci. PubMed Google Scholar, D.C. J. Invest. 67: PubMed Scopus Google Scholar). Our is the first of the of PMN activation on the function and of coronary microvascular endothelial cells. the of PMNs with the endothelium coronary and (10Yuan Y. Mier R.A. Chilian W.M. Zawieja D.C. Granger H.J. Am. J. Physiol. 1995; 268: H490-H498Crossref PubMed Google Scholar). monolayer L.S. Lai L. Malik A.B. J. Cell. Physiol. 1990; 145: 496-500Crossref PubMed Scopus (55) Google Scholar, 13Rosengren S. Olofsson A.M. von Andrian U.H. Lundgren-Akerlund E. Arfors K.E. J. Appl. Physiol. 1991; 71: 1322-1330Crossref PubMed Scopus (52) Google Scholar, C.G. Payne D.K. Mires E. Alexander J.S. J. Biol. Chem. 1998; 273: 15099-15103Abstract Full Text Full Text PDF PubMed Scopus (267) Google Scholar) to to the molecular in cells. with the coronary the monolayer a permeability a to the hyperpermeability of the molecular that to microvascular is large and blood cells to through the endothelial endothelial cells to In this the intercellular junction may conformational the gap formation and macromolecular a transmembrane and β-catenin, a signaling protein that cadherins to the actin cytoskeleton, to in tyrosine phosphorylation concomitant with an increase in suggest that this phosphorylation is an for the AJ and the of cells to Our results and of showed the disappearance of AJ proteins the cell of hyperpermeability (14Carden D. Xiao F. Moak C. Willis B.H. Robinson-Jackson S. Alexander S. Am. J. Physiol. 1998; 275: H385-H392PubMed Google Scholar, 16Del Maschio A. Zanetti A. Corada M. Rival Y. Ruco L. Lampugnani M.G. Dejana E. J. Cell Biol. 1996; 135: 497-510Crossref PubMed Scopus (280) Google Scholar, 17Allport J.R. Ding H. Collins T. Gerritsen M.E. Luscinskas F.W. J. Exp. Med. 1997; 186: 517-527Crossref PubMed Scopus (153) Google Scholar). we observed an increase in the formation of actin stress that with the AJ and permeability In cells stress fiber there is an increase in stress fiber formation across the monolayer control cell monolayers. stress with of the cell membrane and the cell a the between cells. The to protein or protein complex are the actin hyperpermeability Our is that AJ proteins the actin and for the integrity of the monolayer to In to actin stress and with including the AJ to changes in leading to a weak or of VE-cadherin and β-catenin may be a signaling that structural In of data that phosphorylation of VE-cadherin and β-catenin the hyperpermeability in the endothelial by which PMNs increase microvascular permeability has been A of evidence that the is to PMN-derived including and R. T. T. J. Immunol. 1995; PubMed Scopus Google Scholar, M.G. R.A. C. J. Invest. PubMed Scopus Google Scholar, N. A. A. D. J. Exp. 1993; Google Scholar, S. J. Invest. PubMed Scopus Google Scholar). At the site of PMN adhesion, large of the factors are and a of signaling in the endothelium. endothelial cell and intercellular are on and that protein phosphorylation is of the signaling that is activated in to PMN adhesion, and in the hyperpermeability response. In of we that activated PMNs in tyrosine phosphorylation of AJ proteins accompanied by an increase in we that the permeability increase by and a to phosphorylation of β-catenin and VE-cadherin It is that activated PMNs that or both the tyrosine and phosphorylation which may with each Our is that both types of phosphorylation are to hyperpermeability of is the first to PMN activation with phosphorylation of AJ proteins and hyperpermeability in cultured microvascular endothelial monolayers. that tyrosine phosphorylation of the adhesion proteins and adhesion in monolayers in with a of endothelial barrier function Y. Q. J. Am. J. Physiol. 1998; 275: Google Scholar). we have evidence that in both the adhesion complex and the AJ in hyperpermeability of the microvascular endothelium and that activated PMNs of have the for studies the molecular associated with changes in vascular has long been in the of coronary microvascular injury and other types of L.S. Lai L. Malik A.B. J. Cell. Physiol. 1990; 145: 496-500Crossref PubMed Scopus (55) Google Scholar, 13Rosengren S. Olofsson A.M. von Andrian U.H. Lundgren-Akerlund E. Arfors K.E. J. Appl. Physiol. 1991; 71: 1322-1330Crossref PubMed Scopus (52) Google Scholar, J. Cell Biol. 1993; PubMed Scopus Google Scholar, J.S. A. J. Cell Sci. PubMed Google Scholar, J. Cell Sci. PubMed Google Scholar, D.C. J. Invest. 67: PubMed Scopus Google Scholar). Our is the first of the of PMN activation on the function and of coronary microvascular endothelial cells. the of PMNs with the endothelium coronary and (10Yuan Y. Mier R.A. Chilian W.M. Zawieja D.C. Granger H.J. Am. J. Physiol. 1995; 268: H490-H498Crossref PubMed Google Scholar). monolayer L.S. Lai L. Malik A.B. J. Cell. Physiol. 1990; 145: 496-500Crossref PubMed Scopus (55) Google Scholar, 13Rosengren S. Olofsson A.M. von Andrian U.H. Lundgren-Akerlund E. Arfors K.E. J. Appl. Physiol. 1991; 71: 1322-1330Crossref PubMed Scopus (52) Google Scholar, C.G. Payne D.K. Mires E. Alexander J.S. J. Biol. Chem. 1998; 273: 15099-15103Abstract Full Text Full Text PDF PubMed Scopus (267) Google Scholar) to to the molecular in cells. with the coronary the monolayer a permeability a to the hyperpermeability The of the molecular that to microvascular is large and blood cells to through the endothelial endothelial cells to In this the intercellular junction may conformational the gap formation and macromolecular a transmembrane and β-catenin, a signaling protein that cadherins to the actin cytoskeleton, to in tyrosine phosphorylation concomitant with an increase in suggest that this phosphorylation is an for the AJ and the of cells to Our results and of showed the disappearance of AJ proteins the cell of hyperpermeability (14Carden D. Xiao F. Moak C. Willis B.H. Robinson-Jackson S. Alexander S. Am. J. Physiol. 1998; 275: H385-H392PubMed Google Scholar, 16Del Maschio A. Zanetti A. Corada M. Rival Y. Ruco L. Lampugnani M.G. Dejana E. J. Cell Biol. 1996; 135: 497-510Crossref PubMed Scopus (280) Google Scholar, 17Allport J.R. Ding H. Collins T. Gerritsen M.E. Luscinskas F.W. J. Exp. Med. 1997; 186: 517-527Crossref PubMed Scopus (153) Google Scholar). we observed an increase in the formation of actin stress that with the AJ and permeability In cells stress fiber there is an increase in stress fiber formation across the monolayer control cell monolayers. stress with of the cell membrane and the cell a the between cells. The to protein or protein complex are the actin hyperpermeability Our is that AJ proteins the actin and for the integrity of the monolayer to In to actin stress and with including the AJ to changes in leading to a weak or of VE-cadherin and β-catenin may be a signaling that structural In of data that phosphorylation of VE-cadherin and β-catenin the hyperpermeability in the endothelial cells. The by which PMNs increase microvascular permeability has been A of evidence that the is to PMN-derived including and R. T. T. J. Immunol. 1995; PubMed Scopus Google Scholar, M.G. R.A. C. J. Invest. PubMed Scopus Google Scholar, N. A. A. D. J. Exp. 1993; Google Scholar, S. J. Invest. PubMed Scopus Google Scholar). At the site of PMN adhesion, large of the factors are and a of signaling in the endothelium. endothelial cell and intercellular are on and that protein phosphorylation is of the signaling that is activated in to PMN adhesion, and in the hyperpermeability response. In of we that activated PMNs in tyrosine phosphorylation of AJ proteins accompanied by an increase in we that the permeability increase by and a to phosphorylation of β-catenin and VE-cadherin It is that activated PMNs that or both the tyrosine and phosphorylation which may with each Our is that both types of phosphorylation are to hyperpermeability of monolayers. is the first to PMN activation with phosphorylation of AJ proteins and hyperpermeability in cultured microvascular endothelial monolayers. that tyrosine phosphorylation of the adhesion proteins and adhesion in monolayers in with a of endothelial barrier function Y. Q. J. Am. J. Physiol. 1998; 275: Google Scholar). we have evidence that in both the adhesion complex and the AJ in hyperpermeability of the microvascular endothelium and that activated PMNs of have the for studies the molecular associated with changes in