KLF2-dependent, Shear Stress-induced Expression of CD59

Abstract

Complement activation may predispose to vascular injury and atherogenesis. The atheroprotective actions of unidirectional laminar shear stress led us to explore its influence on endothelial cell expression of complement inhibitory proteins CD59 and decay-accelerating factor. Human umbilical vein and aortic endothelial cells were exposed to laminar shear stress (12 dynes/cm2) or disturbed flow (±5 dynes/cm2 at 1Hz) in a parallel plate flow chamber. Laminar shear induced a flow rate-dependent increase in steady-state CD59 mRNA, reaching 4-fold at 12 dynes/cm2. Following 24–48 h of laminar shear stress, cell surface expression of CD59 was up-regulated by 100%, whereas decay-accelerating factor expression was unchanged. The increase in CD59 following laminar shear was functionally significant, reducing C9 deposition and complement-mediated lysis of flow-conditioned endothelial cells by 50%. Although CD59 induction was independent of PI3-K, ERK1/2 and nitric oxide, an RNA interference approach demonstrated dependence upon an ERK5/KLF2 signaling pathway. In contrast to laminar shear stress, disturbed flow failed to induce endothelial cell CD59 protein expression. Likewise, CD59 expression on vascular endothelium was significantly higher in atheroresistant regions of the murine aorta exposed to unidirectional laminar shear stress, when compared with atheroprone areas exposed to disturbed flow. We propose that up-regulation of CD59 via ERK5/KLF2 activation leads to endothelial resistance to complement-mediated injury and protects from atherogenesis in regions of laminar shear stress. Complement activation may predispose to vascular injury and atherogenesis. The atheroprotective actions of unidirectional laminar shear stress led us to explore its influence on endothelial cell expression of complement inhibitory proteins CD59 and decay-accelerating factor. Human umbilical vein and aortic endothelial cells were exposed to laminar shear stress (12 dynes/cm2) or disturbed flow (±5 dynes/cm2 at 1Hz) in a parallel plate flow chamber. Laminar shear induced a flow rate-dependent increase in steady-state CD59 mRNA, reaching 4-fold at 12 dynes/cm2. Following 24–48 h of laminar shear stress, cell surface expression of CD59 was up-regulated by 100%, whereas decay-accelerating factor expression was unchanged. The increase in CD59 following laminar shear was functionally significant, reducing C9 deposition and complement-mediated lysis of flow-conditioned endothelial cells by 50%. Although CD59 induction was independent of PI3-K, ERK1/2 and nitric oxide, an RNA interference approach demonstrated dependence upon an ERK5/KLF2 signaling pathway. In contrast to laminar shear stress, disturbed flow failed to induce endothelial cell CD59 protein expression. Likewise, CD59 expression on vascular endothelium was significantly higher in atheroresistant regions of the murine aorta exposed to unidirectional laminar shear stress, when compared with atheroprone areas exposed to disturbed flow. We propose that up-regulation of CD59 via ERK5/KLF2 activation leads to endothelial resistance to complement-mediated injury and protects from atherogenesis in regions of laminar shear stress. The complement cascade provides an essential defense against bacterial infection and a bridge between innate and adaptive immunity (1Walport M.J. N. Engl. J. Med. 2001; 344: 1058-1066Crossref PubMed Scopus (2427) Google Scholar). However, by the nature of its cytolytic activity, complement has the potential to inflict injury on bystander host tissues including vascular endothelium. C1q, C3a, C5a, and the C5b-9 membrane attack complex (MAC) 3The abbreviations used are: MAC, membrane attack complex; EC, endothelial cells; DAF, decay-accelerating factor; ERK, extracellular signal-regulated kinase; KLF2, Kruppel-like factor 2; LSS, laminar shear stress; DF, disturbed flow; PI3-K, phosphoinositide 3-kinase; l-NMMA, NG-monomethyl-l-arginine; HUVEC, human umbilical vein endothelial cells; HAEC, human aortic endothelial cells; qrt, quantitative real time polymerase chain reaction; NHS, normal human serum; mAb, monoclonal antibody; MCP, membrane cofactor protein; siRNA, short interfering RNA; eNOS, endothelial nitricoxide synthase. have the capacity to exert pro-inflammatory effects on vascular endothelial cells (EC) including induction of cellular adhesion molecules and cytokine secretion, increased leukocyte adhesion, and generation of a pro-thrombotic endothelial surface (2Lozada C. Levin R.I. Huie M. Hirschhorn R. Naime D. Whitlow M. Recht P.A. Golden B. Cronstein B.N. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8378-8382Crossref PubMed Scopus (126) Google Scholar, 3Saadi S. Holzknecht R.A. Patte C.P. Stern D.M. Platt J.L. J. Exp. Med. 1995; 182: 1807-1814Crossref PubMed Scopus (250) Google Scholar, 4Tedesco F. Pausa M. Nardon E. Introna M. Mantovani A. Dobrina A. J. Exp. Med. 1997; 185: 1619-1627Crossref PubMed Scopus (256) Google Scholar, 5Albrecht E.A. Chinnaiyan A.M. Varambally S. Kumar-Sinha C. Barrette T.R. Sarma J.V. Ward P.A. Am. J. Pathol. 2004; 164: 849-859Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar). Mechanisms implicated in complement deposition on the surface of EC include activation of the classical pathway by immune complexes, anti-phospholipid, and anti-endothelial cell antibodies (6Fleming S.D. Egan R.P. Chai C. Girardi G. Holers V.M. Salmon J. Monestier M. Tsokos G.C. J. Immunol. 2004; 173: 7055-7061Crossref PubMed Scopus (78) Google Scholar) and through recognition of apoptotic cell blebs by the globular head of C1q (7Navratil J.S. Watkins S.C. Wisnieski J.J. Ahearn J.M. J. Immunol. 2001; 166: 3231-3239Crossref PubMed Scopus (213) Google Scholar). Alternatively, activation of the lectin pathway may follow exposure to hypoxia reoxygenation (8Collard C.D. Vakeva A. Morrissey M.A. Agah A. Rollins S.A. Reenstra W.R. Buras J.A. Meri S. Stahl G.L. Am. J. Pathol. 2000; 156: 1549-1556Abstract Full Text Full Text PDF PubMed Scopus (301) Google Scholar). Complement activation on the surface of human EC is regulated by membrane-bound inhibitory proteins: decay-accelerating factor (DAF) (CD55), membrane cofactor protein (MCP, CD46), and CD59. The genes encoding DAF and MCP are clustered on the long arm of chromosome 1, whereas that for CD59 is located on chromosome 11. These proteins use distinct mechanisms for complement regulation. DAF prevents the formation and accelerates the decay of C3 and C5 convertases (9Lublin D.M. Atkinson J.P. Annu. Rev. Immunol. 1989; 7: 35-58Crossref PubMed Scopus (401) Google Scholar), whereas MCP accelerates the degradation of C3b and C4b by Factor I (10Liszewski M.K. Post T.W. Atkinson J.P. Annu. Rev. Immunol. 1991; 9: 431-455Crossref PubMed Scopus (501) Google Scholar). CD59 inhibits the terminal pathway of complement activation, preventing incorporation of C9 into the MAC (11Davies A. Simmons D.L. Hale G. Harrison R.A. Tighe H. Lachmann P.J. Waldmann H. J. Exp. Med. 1989; 170: 637-654Crossref PubMed Scopus (538) Google Scholar, 12Okada N. Harada R. Fujita T. Okada H. Int. Immunol. 1989; 1: 205-208Crossref PubMed Scopus (172) Google Scholar). Complement activation may be an early prelesional event in atherogenesis, as revealed by colocalization of C5b-9 with lipid deposits in the tunica intima prior to monocyte recruitment (13Seifert P.S. Hugo F. Hansson G.K. Bhakdi S. Lab. Investig. 1989; 60: 747-754PubMed Google Scholar). Experimental models suggest that vascular endothelial injury is the earliest detectable event in atherogenesis. Apoptosis of EC occurs preferentially at bifurcations and curvatures, where denudation of vascular endothelium enhances the risk of plaque development and local thrombosis. Moreover, aging and exposure to oxidized low density lipoprotein or reactive oxygen species increases EC apoptosis, consistent with a role in the initiation of atherogenesis (14Dimmeler S. Haendeler J. Zeiher A.M. Curr. Opin. Lipidol. 2002; 13: 531-536Crossref PubMed Scopus (80) Google Scholar). In addition to local lipid-related activation of complement, exposure of the underlying extracellular matrix, resulting from apoptosis of EC, may further reinforce complement activation (13Seifert P.S. Hugo F. Hansson G.K. Bhakdi S. Lab. Investig. 1989; 60: 747-754PubMed Google Scholar, 15Hindmarsh E.J. Marks R.M. J. Immunol. 1998; 160: 6128-6136PubMed Google Scholar). The susceptibility of branch points and curvatures to atherosclerosis is in large part related to exposure to disturbed blood flow (DF), with low shear reversing or oscillatory flow patterns. In contrast, the arterial tree exposed to unidirectional laminar shear stress (LSS) >10 dynes/cm2 tends to be protected (16Cunningham K.S. Gotlieb A.I. Lab. Investig. 2005; 85: 9-23Crossref PubMed Scopus (837) Google Scholar). This is reflected in the phenotype of EC exposed to LSS, typically characterized by enhanced NO biosynthesis, prolonged cell survival, and an anticoagulant, anti-adhesive cell surface (17Dimmeler S. Hermann C. Galle J. Zeiher A.M. Arterioscler. Thromb. Vasc. Biol. 1999; 19: 656-664Crossref PubMed Scopus (271) Google Scholar, 18Sheikh S. Rainger G. Gale Z. Rahman M. Nash G. Blood. 2003; 102: 2828-2834Crossref PubMed Scopus (113) Google Scholar, 19Yamawaki H. Lehoux S. Berk B.C. Circulation. 2003; 108: 1619-1625Crossref PubMed Scopus (153) Google Scholar). In contrast, endothelium exposed to DF exhibits reduced levels of eNOS, increased apoptosis, generation of reactive oxygen species, permeability to low density lipoprotein, and leukocyte adhesion (reviewed in Ref. 16Cunningham K.S. Gotlieb A.I. Lab. Investig. 2005; 85: 9-23Crossref PubMed Scopus (837) Google Scholar). The importance of unidirectional LSS for vascular endothelial cytoprotection, together with the observation that LSS protects against complement-induced EC activation and chemokine synthesis (20Urbich C. Fritzenwanger M. Zeiher A.M. Dimmeler S. Circulation. 2000; 101: 352-355Crossref PubMed Scopus (52) Google Scholar), led us to explore whether LSS may be protective against complement-mediated vascular injury through suppression of the C5b-9 membrane attack complex. We show for the first time that CD59, the predominant membrane-bound regulator of the MAC, is preferentially induced by unidirectional atheroprotective LSS, providing enhanced protection against complement activation. This response was independent of NO and dependent upon an ERK5, Kruppel-like factor 2 (KLF2) signaling pathway. In contrast, an atheroprone-disturbed flow waveform failed to increase CD59 protein expression. These observations were confirmed in vivo, where CD59 expression was significantly higher on arterial EC in atherosclerosis-resistant areas of the murine aorta. Thus, up-regulation of CD59 may represent an important component of endothelial cytoprotection in regions of LSS, preserving vascular integrity and minimizing complement activation and atherogenesis. CD59 was from the The CD59 was a from of of and were from D. and J. of and were from and was from were from to umbilical vein and human aortic from were as Z. R. E.A. A. A.M. 2002; PubMed Scopus Google Scholar). The use of human EC was by or were exposed to shear unidirectional laminar flow to or disturbed flow with of flow at dynes/cm2) for to h a parallel plate flow as R.M. R.M. 1998; PubMed Scopus Google Scholar, J. H. H. M. A. M. R. J. PubMed Scopus Google Scholar). was by of EC contrast cell and of and short interfering RNA were from or for were and for were and were were at on in endothelial cell to KLF2, ERK5, or at was into EC in endothelial cell EC were for h in endothelial cell and for expression by or The of was confirmed a of was an as R. M. E.A. PubMed Scopus Google Scholar, F. J.J. E.A. T. A.M. J. Thromb. PubMed Scopus Google Scholar). and were used as with in to and with and RNA was and to the was in a of of and and and used KLF2, KLF2, CD59, CD59, ERK5, and ERK5, was from the RNA was on a to and by to a for human CD59 from H. of as H. Blood. 1999; PubMed Google Scholar). density for were with an with to the on and as from was as a flow H. Blood. 1999; PubMed Google Scholar). The are as the the with by the an Complement surface C9 deposition and complement-mediated lysis was by flow as R. M. E.A. PubMed Scopus Google Scholar). EC were exposed to LSS at 12 dynes/cm2 for and in The inhibitory CD59 J. Immunol. Google Scholar) was used at of C9 EC were exposed to normal human or for to h at C9 was with and cell lysis was by the of cells to by flow following exposure to or for and was by of an S. J.J. Am. J. Pathol. 170: Full Text Full Text PDF PubMed Scopus Google Scholar). were by and the with in were and in for The aortic was and a was that the of the aorta at the of the the and the of its and The was in and were and with normal by CD59 for The were and with and The used for were for CD59. The were with as and with by were with by and with monoclonal the the were in and for with and in were with a and were to for was detectable between However, to of by was on was with and by of the into J. The regions of were and the was used to the of on the to were from and in with and were of the were and to were and for a of murine are as the of The were to and and the of with or an was used to in regions of the murine aorta an The were at of CD59 by the influence of shear stress on the surface expression of the complement inhibitory proteins MCP, DAF, and CD59, in a parallel plate flow were exposed to unidirectional LSS (12 dynes/cm2) for to h and by flow in the cell surface expression of MCP or DAF was In contrast, a increase in CD59 expression of to was following exposure to unidirectional LSS for and was at h of CD59 R. M. E.A. PubMed Scopus Google and demonstrated that induction by LSS was with an increase in mRNA, first detectable 2 h the of flow and to This was confirmed by revealed a increase of to 24–48 h of LSS whether up-regulation was dependent upon the of shear were exposed to LSS for and in CD59 were by CD59 levels as LSS increased to 12 dynes/cm2 on to represent EC from a vascular by atherosclerosis a up-regulation of CD59 and cell surface protein in response to LSS The extracellular upon EC are may influence to shear stress J.M. M. E. M.A. J. Biol. 2005; PubMed Scopus Google Scholar). were on and I and exposed to LSS for However, the nature of the underlying the of LSS to induce CD59, with induction CD59 the of CD59 the of LSS on cell surface deposition of C9 was were or in the of LSS (12 dynes/cm2) for h prior to exposure to and flow an against a on C9 that is revealed upon C5b-9 complex In complement-mediated EC lysis was R. M. E.A. PubMed Scopus Google Scholar). to in C9 deposition on the surface of in C9 deposition was on EC exposed to LSS with levels with that on EC exposed to that to complement activation Likewise, LSS protection against complement-mediated lysis when compared with EC The CD59 J. Immunol. Google Scholar) was used to the role of CD59 in protection against the protective of LSS, and its in lysis of of EC of of signaling as regulated by and ERK1/2 and enhanced NO may to the effects of LSS G. Berk B.C. 13: PubMed Scopus Google Scholar). Thus, a approach to explore the role of in the induction of CD59 by were with or at have to of and E.A. R. M. A. Am. J. 2005; PubMed Scopus Google Scholar, R. E.A. J.J. M. A.M. M. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). of inhibitory on the induction of CD59 in response to LSS Although reduced Likewise, the of nitricoxide l-NMMA, at of the induction of CD59 by R. M. E.A. PubMed Scopus Google Scholar), CD59 protein or expression In contrast, of the protein synthesis the increase in CD59 dependence upon synthesis of an protein in response to has that in addition to may S. T. E. J. Biol. 1999; Full Text Full Text PDF PubMed Scopus Google the in CD59 in the of may in a in activation. These with the of as a of the C. Berk B.C. 2004; PubMed Scopus Google Scholar), of the factor as a of D. A. Biol. 2005; PubMed Scopus Google Scholar, G. Z. M.K. M.A. G. J. Investig. PubMed Scopus Google Scholar), and of may increase CD59 in EC G. Z. M.K. M.A. G. J. Investig. PubMed Scopus Google Scholar), led us to the role of ERK5/KLF2 The are a of the has as an important factor in the of endothelial S. Z. D.M. R.M. A. Z. M.A. G. M.K. J. Exp. Med. 2004; PubMed Scopus Google Scholar, R.A. A. J. H. Blood. PubMed Scopus Google Scholar). We an reduced the expression of in EC by demonstrated that the induction of CD59 and surface protein by LSS was significantly in with siRNA, when compared with LSS significantly induced expression of mRNA, a response that was by the of In of significantly induction of by LSS of CD59 in with demonstrated that LSS induction of CD59 was an that was with This was further confirmed by flow that cell surface expression of CD59 on EC with and exposed to LSS was to that on cells CD59 by LSS and and atheroprone regions of the aorta are exposed to unidirectional LSS and a low reversing flow the of flow on CD59 EC were exposed to LSS (12 or an oscillatory flow (±5 dynes/cm2 at to in endothelial cell and CD59 expression were compared with cells to the demonstrated in response to LSS and induction of adhesion by LSS and vascular cell adhesion by DF, R.M. 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Biol. 1998; PubMed Scopus Google Scholar) may be in the of atherosclerosis from the prelesional (13Seifert P.S. Hugo F. Hansson G.K. Bhakdi S. Lab. Investig. 1989; 60: 747-754PubMed Google Scholar), through early arterial M. J. J. C. Arterioscler. Thromb. Vasc. Biol. 1997; PubMed Scopus Google Scholar) and and R. F. A. Full Text PDF PubMed Scopus Google Scholar), via generation of C3a, C5a, and the C5b-9 LSS is an essential component of vascular endothelial to resistance against apoptosis and the of an anti-adhesive endothelial (reviewed in Ref. Berk B.C. Arterioscler. Thromb. Vasc. Biol. 1998; PubMed Scopus Google Scholar). Although induction of and NO is an important regulator of of mechanisms (17Dimmeler S. Hermann C. Galle J. Zeiher A.M. Arterioscler. Thromb. Vasc. Biol. 1999; 19: 656-664Crossref PubMed Scopus (271) Google Scholar), the effects of LSS to be CD59 is a to the of cell surface proteins (11Davies A. Simmons D.L. Hale G. Harrison R.A. Tighe H. Lachmann P.J. Waldmann H. J. Exp. Med. 1989; 170: 637-654Crossref PubMed Scopus (538) Google Scholar, 12Okada N. Harada R. Fujita T. Okada H. Int. Immunol. 1989; 1: 205-208Crossref PubMed Scopus (172) Google Scholar). its to to the of CD59 prevents the incorporation of C9 into C5b-9 R. Okada N. Fujita T. Okada H. J. Immunol. Google Scholar). Thus, CD59, is on the vascular endothelial is the predominant membrane-bound regulator of the In of the role of complement activation in atherogenesis and of LSS in the of shear stress on endothelial cell CD59 expression. We have that 24–48 h of atheroprotective LSS enhances CD59 protein expression in and and that response is in EC exposed to an oscillatory flow (±5 dynes/cm2 at disturbed flow of atheroprone regions of the of the of CD59 expression at of the murine aorta exposed to LSS and DF the of observations to the in Moreover, the enhanced expression of CD59 in response to LSS in increased endothelial resistance to C5b-9 deposition and complement-mediated (20Urbich C. 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