Endothelial Cell Overexpression of Fas Ligand Attenuates Ischemia-Reperfusion Injury in the Heart
2003; Elsevier BV; Volume: 278; Issue: 17 Linguagem: Inglês
10.1074/jbc.m211707200
ISSN1083-351X
AutoresYang Jiang, Steven P. Jones, Toshimitsu Suhara, James J.M. Greer, Paul D. Ware, Nhan P. Nguyen, Harris Perlman, David P. Nelson, David J. Lefer, Kenneth Walsh,
Tópico(s)Immune Response and Inflammation
ResumoFas ligand (FasL) is a member of tumor necrosis factor family that induces apoptosis in target cells that express Fas. The function of FasL during inflammation remains controversial. In this study, we examined the role of vascular endothelial FasL during acute myocardial ischemia-reperfusion that is closely associated with inflammation. Transgenic mouse lines were established that overexpress human FasL on endothelium under the control of the vascular endothelial cadherin promoter. Expression of FasL transgene was detected at both mRNA and protein levels, and functional transgene-encoded FasL protein was specifically expressed on the surface of vascular endothelial cells. Transgenic mice developed normally and had normal hearts. When subjected to 30 min of myocardial ischemia and 72 h of reperfusion, myocardial infarct size was reduced by 42% in the transgenic mice compared with nontransgenic littermates (p < 0.05). Moreover, hemodynamic data demonstrated that transgenic hearts performed better following ischemia and reperfusion compared with nontransgenic hearts. Myocardial neutrophil infiltration was reduced by 54% after 6 h of reperfusion in transgenic hearts (p < 0.01). Neutrophil depletion prior to ischemia-reperfusion injury led to smaller infarcts that were not different between transgenic and nontransgenic mice, suggesting that endothelial FasL may attenuate ischemia-reperfusion injury by abating the inflammatory response. These results indicate that vascular endothelial FasL may exert potent anti-inflammatory actions in the setting of myocardial ischemia-reperfusion injury. Fas ligand (FasL) is a member of tumor necrosis factor family that induces apoptosis in target cells that express Fas. The function of FasL during inflammation remains controversial. In this study, we examined the role of vascular endothelial FasL during acute myocardial ischemia-reperfusion that is closely associated with inflammation. Transgenic mouse lines were established that overexpress human FasL on endothelium under the control of the vascular endothelial cadherin promoter. Expression of FasL transgene was detected at both mRNA and protein levels, and functional transgene-encoded FasL protein was specifically expressed on the surface of vascular endothelial cells. Transgenic mice developed normally and had normal hearts. When subjected to 30 min of myocardial ischemia and 72 h of reperfusion, myocardial infarct size was reduced by 42% in the transgenic mice compared with nontransgenic littermates (p < 0.05). Moreover, hemodynamic data demonstrated that transgenic hearts performed better following ischemia and reperfusion compared with nontransgenic hearts. Myocardial neutrophil infiltration was reduced by 54% after 6 h of reperfusion in transgenic hearts (p < 0.01). Neutrophil depletion prior to ischemia-reperfusion injury led to smaller infarcts that were not different between transgenic and nontransgenic mice, suggesting that endothelial FasL may attenuate ischemia-reperfusion injury by abating the inflammatory response. These results indicate that vascular endothelial FasL may exert potent anti-inflammatory actions in the setting of myocardial ischemia-reperfusion injury. Fas ligand tumor necrosis factor reverse transcriptase transgenic nontransgenic fluorescence-activated cell sorter atrial natriuretic factor phospholamban left ventricle phosphate-buffered saline myocardial ischemia-reperfusion injury infarction area at risk myosin heavy chain vascular endothelial cadherin left anterior descending polymorphonuclear leukocytes Serca2a Fas ligand (FasL)1 is a type II membrane protein belonging to the tumor necrosis factor (TNF) family. It induces apoptosis in various cell types that bear the receptor Fas (also called APO-1 or CD95) (1Nagata S. Golstein P. Science. 1995; 267: 1449-1456Crossref PubMed Scopus (3965) Google Scholar). FasL and Fas-deficient mice, gld and lpr mice, respectively, develop similar phenotypes including splenomegaly, lymphadenopathy, and the accumulation of autoantibodies (2Cohen P.L. Eisenberg R.A. Annu. Rev. Immunol. 1991; 9: 243-269Crossref PubMed Scopus (1131) Google Scholar). These phenotypes suggest that this receptor/ligand pair functions to regulate immune cell homeostasis. Cells differ markedly in their sensitivity to Fas-mediated apoptosis due in part to differences in the expression of FLIP, an inhibitor of Fas-mediated death signaling (3Tschopp J. Irmler M. Thome M. Curr. Opin. Immunol. 1998; 10: 552-558Crossref PubMed Scopus (467) Google Scholar). Endothelial cells express high levels of FLIP and are relatively resistant to FasL-induced apoptosis (4Sata M. Suhara T. Walsh K. Arterioscler. Thromb. Vasc. Biol. 2000; 20: 309-316Crossref PubMed Scopus (111) Google Scholar, 5Suhara T. Mano T. Oliveira B.E. Walsh K. Circ. Res. 2001; 89: 13-19Crossref PubMed Scopus (164) Google Scholar, 6Sata M. Walsh K. J. Biol. Chem. 1998; 273: 33103-33106Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar). In contrast, cardiac myocytes will undergo apoptotic cell death when presented with membrane-bound FasL (7Lee P. Sata M. Lefer D.J. Factor S.M. Walsh K. Kitsis R.N. Am. J. Physiol. 2003; 284: H456-H463Crossref PubMed Scopus (29) Google Scholar). Furthermore, FLIP levels are down-regulated in ischemic regions of the heart following reperfusion (8Rasper D.M. Vaillancourt J.P. Hadano S. Houtzager V.M. Seiden I. Keen S.L.C. Tawa P. Xanthoudakis S. Nasir J. Martindale D. Koop B.F. Peterson E.P. Thornberry N.A. Huang J.Q. Macpherson D.P. Black S.C. Hornung F. Lenardo M.J. Hayden M.R. Roy S. Nicholson D.W. Cell Death Differ. 1998; 5: 271-288Crossref PubMed Scopus (279) Google Scholar), suggesting that Fas-mediated apoptosis may contribute to tissue damage under these conditions.The role of FasL in inflammation has received considerable attention. FasL expression is detected at some immune privileged sites, for example, eye and testis (9Griffith T.S. Brunner T. Fletcher S.M. Green D.R. Ferguson T.A. Science. 1995; 270: 1189-1192Crossref PubMed Scopus (1863) Google Scholar, 10Bellgrau D. Gold D. Selawry H. Moore J. Franzusoff A. Duke R.C. Nature. 1995; 377: 630-632Crossref PubMed Scopus (1097) Google Scholar), where it is believed to protect those tissues by inducing apoptosis in the intruding Fas-bearing immune cells (9Griffith T.S. Brunner T. Fletcher S.M. Green D.R. Ferguson T.A. Science. 1995; 270: 1189-1192Crossref PubMed Scopus (1863) Google Scholar). Although some studies have shown that FasL expression can promote allograft survival in transplantation models, other studies have reported that ectopic FasL promotes neutrophil infiltration and accelerates rejection (reviewed in Ref. 11Green D.R. Ferguson T.A. Nat. Rev. Mol. Cell. Biol. 2001; 2: 917-924Crossref PubMed Scopus (214) Google Scholar). FasL-induced inflammatory responses can result from the release of interleukin-1β (12Miwa K. Asano M. Horai R. Iwakura Y. Nagata S. Suda T. Nat. Med. 1998; 4: 1287-1292Crossref PubMed Scopus (344) Google Scholar) and other cytokines that recruit neutrophils (13Hohlbaum A.M. Gregory M.S. Ju S.T. Marshak-Rothstein A. J. Immunol. 2001; 167: 6217-6224Crossref PubMed Scopus (136) Google Scholar). In this regard, it has been shown that ectopic FasL expression in cardiac myocytes promotes proinflammatory cytokine expression, leukocyte infiltration, and hypertrophy (14Nelson D.P. Setser E. Hall D.G. Schwartz S.M. Hewitt T. Klevitsky R. Osinska H. Bellgrau D. Duke R.C. Robbins J. J. Clin. Invest. 2000; 105: 1199-1208Crossref PubMed Scopus (75) Google Scholar). Heart grafts overexpressing FasL in cardiac myocytes also undergo accelerated rejection, accompanied by massive neutrophil infiltration (15Takeuchi T. Ueki T. Nishimatsu H. Kajiwara T. Ishida T. Jishage K. Ueda O. Suzuki H. Li B. Moriyama N. Kitamura T. J. Immunol. 1999; 162: 518-522PubMed Google Scholar).Inflammatory reactions involving leukocytes have been shown to contribute to the pathogenesis of myocardial ischemia-reperfusion injury (MI/R) (16Frangogiannis N.G. Smith C.W. Entman M.L. Cardiovasc. Res. 2002; 53: 31-47Crossref PubMed Scopus (1704) Google Scholar). Necrotic cardiomyocytes release cytokines causing leukocytes to transmigrate through the endothelium and accumulate at the ischemic site. Once in the myocardium, leukocytes release proteolytic enzymes and reactive oxygen species, causing further damage to the heart. Neutrophils play a critical role in reperfusion injury, and it has been shown that neutrophil depletion results in reduced myocardial infarct size (17Litt M.R. Jeremy R.W. Weisman H.F. Winkelstein J.A. Becker L.C. Circulation. 1989; 80: 1816-1827Crossref PubMed Scopus (301) Google Scholar, 18Romson J.L. Hook B.G. Kunkel S.L. Abrams G.D. Schork M.A. Lucchesi B.R. Circulation. 1983; 67: 1016-1023Crossref PubMed Scopus (1028) Google Scholar).Besides its expression on immune cells, FasL is also expressed at low levels on the vascular endothelium (4Sata M. Suhara T. Walsh K. Arterioscler. Thromb. Vasc. Biol. 2000; 20: 309-316Crossref PubMed Scopus (111) Google Scholar). Administration of the pro-inflammatory cytokine TNF-α down-regulates FasL expression in endothelial cells, and overexpression of FasL on the endothelium attenuates leukocyte extravasation induced by TNF-α (19Sata M. Walsh K. Nat. Med. 1998; 4: 415-420Crossref PubMed Scopus (208) Google Scholar). It has also been shown that FasL overexpression on the vascular endothelium of carotid arteries inhibits transplant-associated intimal hyperplasia (20Sata M. Luo Z. Walsh K. J. Immunol. 2001; 166: 6964-6971Crossref PubMed Scopus (50) Google Scholar) and that adenovirus-mediated delivery of FasL to injured vessels inhibits intimal hyperplasia in animals with preexisting immunity to adenovirus (21Luo Z. Sata M. Nguyen T. Kaplan J.M. Akita G.Y. Walsh K. Circulation. 1999; 99: 1776-1779Crossref PubMed Scopus (79) Google Scholar, 22Sata M. Perlman H. Muruve D.A. Silver M. Ikebe M. Libermann T.A. Oettgen P. Walsh K. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 1213-1217Crossref PubMed Scopus (135) Google Scholar). These data suggest that when expressed in endothelial cells, FasL may have anti-inflammatory actions.To elucidate further the function of FasL in endothelium, we constructed transgenic mouse lines that stably express FasL in vascular endothelium under the control of the vascular endothelial cadherin (VEcadherin) promoter. When subjected to ischemia-reperfusion, the hearts of VEcadFasL transgenic mice displayed significantly smaller infarct size and a reduction in functional impairment when compared with nontransgenic mice. Consistent with these data, a decrease in neutrophil activity was observed at an early time point in transgenic hearts compared with nontransgenic hearts. No differences in infarct size were seen between transgenic and nontransgenic mice that were treated with cytotoxic anti-neutrophil serum prior to injury. These data support the hypothesis that endothelial FasL acts to minimize neutrophil extravasation and organ damage following ischemia-reperfusion injury.DISCUSSIONIn this study we described the effects of endothelial FasL overexpression on myocardial damage following ischemia-reperfusion injury. We established a new mouse model that specifically expresses FasL in vascular endothelial cells. These mice developed normally and had normal hearts but exhibited a 42% reduction in infarct size following myocardial ischemia-reperfusion injury. Consistent with the smaller infarct size, VEcadFasL mouse hearts displayed less functional impairment than nontransgenic mice following the injury. The cardioprotective effect in the transgenic mice may be attributable to a 54% reduction in neutrophil accumulation in the reperfused tissue. Thus, overexpression of FasL on vascular endothelium may serve to minimize neutrophil recruitment, thereby reducing myocardial damage.A number of studies have analyzed the role of the Fas/FasL system in the heart. The α-MyHC-FasL transgenic mice that ectopically express FasL in cardiac myocytes develop mild cardiac hypertrophy associated with interstitial fibrosis and leukocyte inflammation (14Nelson D.P. Setser E. Hall D.G. Schwartz S.M. Hewitt T. Klevitsky R. Osinska H. Bellgrau D. Duke R.C. Robbins J. J. Clin. Invest. 2000; 105: 1199-1208Crossref PubMed Scopus (75) Google Scholar). Although apoptosis was not observed in the α-MyHC-FasL hearts, other experiments have provided direct evidence that Fas pathway can induce apoptosis in cardiomyocytes. In the study of Lee et al. (7Lee P. Sata M. Lefer D.J. Factor S.M. Walsh K. Kitsis R.N. Am. J. Physiol. 2003; 284: H456-H463Crossref PubMed Scopus (29) Google Scholar), it was shown that FasL expression promotes cardiomyocyte apoptosis bothin vivo and in vitro and that Fas-deficientlpr mice displayed less cardiomyocyte apoptosis and correspondingly smaller infarct size after ischemia-reperfusion injury. Hearts from lpr mice also display fewer apoptotic cellsex vivo after ischemia-reperfusion (31Jeremias I. Kupatt C. Martin-Villalba A. Habazettl H. Schenkel J. Boekstegers P. Debatin K.M. Circulation. 2000; 102: 915-920Crossref PubMed Scopus (192) Google Scholar), and intravenous administration of FasL-blocking antibody attenuates inflammation in the heart and reduces myocardial infarct size following ischemia-reperfusion injury (32Shiraishi H. Toyozaki T. Tsukamoto Y. Saito T. Masuda Y. Hiroshima K. Ohwada H. Kobayashi N. Hiroe M. Lab. Invest. 2002; 82: 1121-1129Crossref PubMed Scopus (13) Google Scholar). It is possible that the inflammation and hypertrophy in α-MyHC-FasL mouse hearts result from chronic, but undetectably low, levels of apoptosis in cardiac myocytes or tissue-resident inflammatory cells (13Hohlbaum A.M. Gregory M.S. Ju S.T. Marshak-Rothstein A. J. Immunol. 2001; 167: 6217-6224Crossref PubMed Scopus (136) Google Scholar). Alternatively, the cardiac hypertrophy observed in α-MyHC-FasL mice may result from an activation of intrinsic growth regulatory signaling cascades that promote the phosphorylation of glycogen synthase kinase 3β within cardiac myocytes (33Badorff C. Ruetten H. Mueller S. Stahmer M. Gehring D. Jung F. Ihling C. Zeiher A.M. Dimmeler S. J. Clin. Invest. 2002; 109: 373-381Crossref PubMed Scopus (145) Google Scholar). In this regard, Fas is reported to positively affect the growth of a number of cell types (reviewed in Ref. 34Budd R.C. J. Clin. Invest. 2002; 109: 437-441Crossref PubMed Scopus (131) Google Scholar); however, cell cycle entry in some of these cases may result from the execution of the apoptotic process (for example see Ref. 35Perlman H. Sata M. Krasinski K. Dorai T. Buttyan R. Walsh K. Cardiovasc. Res. 2000; 45: 570-578Crossref PubMed Scopus (42) Google Scholar).In contrast to cardiomyocytes, vascular endothelial cells express FasL, and overexpression of FasL in these cells normally does not affect viability or cell cycle status (4Sata M. Suhara T. Walsh K. Arterioscler. Thromb. Vasc. Biol. 2000; 20: 309-316Crossref PubMed Scopus (111) Google Scholar). Thus, the overexpression of FasL by the endothelial cells of transgenic mice would be expected to alter immune responses in the absence of cytotoxic or proliferative effects on the endothelium. The marked differences between the heart phenotypes of VEcadFasL and α-MyHC-FasL mice clearly illustrate that FasL can exert either pro-inflammatory or anti-inflammatory actions within an organ depending upon differences in cell type-specific expression or microenvironment. Ectopic expression of FasL in cardiac myocytes leads to inflammation and tissue damage, whereas overexpression of FasL on the vascular endothelium has an anti-inflammatory effect and protects the heart from injury. Similar to our findings in heart, both pro- and anti-inflammatory effects of FasL expression have also been observed in other transgenic models. Transgenic mice expressing FasL in pancreatic beta cells display neutrophil infiltration and develop diabetes at a young age (36Kang S.M. Schneider D.B. Lin Z. Hanahan D. Dichek D.A. Stock P.G. Baekkeskov S. Nat. Med. 1997; 3: 738-743Crossref PubMed Scopus (428) Google Scholar). In this setting, ectopic expression of FasL will lead to the destruction of Fas-positive islet cells (37Muruve D.A. Nicolson A.G. Manfro R.C. Strom T.B. Sukhatme V.P. Libermann T.A. Hum. Gene Ther. 1997; 8: 955-963Crossref PubMed Scopus (69) Google Scholar). In contrast, FasL is expressed on follicular cells of the thyroid (38Stassi G. Di Liberto D. Todaro M. Zeuner A. Ricci-Vitiani L. Stoppacciaro A. Ruco L. Farina F. Zummo G. De Maria R. Nat. Immunol. 2000; 1: 483-488Crossref PubMed Scopus (133) Google Scholar), and transgenic expression of FasL on thyroid follicular cells prevents thyroid allograft rejection (39Tourneur L. Malassagne B. Batteux F. Fabre M. Mistou S. Lallemand E. Lores P. Chiocchia G. J. Immunol. 2001; 167: 1338-1346Crossref PubMed Scopus (31) Google Scholar). These data support the hypothesis that FasL-induced inflammation involves FasL that is expressed ectopically, whereas anti-inflammatory effects are associated with overexpression in cell types that normally express endogenous FasL (11Green D.R. Ferguson T.A. Nat. Rev. Mol. Cell. Biol. 2001; 2: 917-924Crossref PubMed Scopus (214) Google Scholar).Neutrophils accumulate at the site of ischemia within hours of reperfusion and damage the tissue by releasing proteases and oxidants (40Dreyer W.J. Michael L.H. West M.S. Smith C.W. Rothlein R. Rossen R.D. Anderson D.C. Entman M.L. Circulation. 1991; 84: 400-411Crossref PubMed Scopus (258) Google Scholar). It is well established that neutrophil depletion will reduce ischemia-reperfusion injury in the heart (17Litt M.R. Jeremy R.W. Weisman H.F. Winkelstein J.A. Becker L.C. Circulation. 1989; 80: 1816-1827Crossref PubMed Scopus (301) Google Scholar, 18Romson J.L. Hook B.G. Kunkel S.L. Abrams G.D. Schork M.A. Lucchesi B.R. Circulation. 1983; 67: 1016-1023Crossref PubMed Scopus (1028) Google Scholar). Neutrophils express both Fas and FasL and are susceptible to Fas-mediated cell death (41Liles W.C. Kiener P.A. Ledbetter J.A. Aruffo A. Klebanoff S.J. J. Exp. Med. 1996; 184: 429-440Crossref PubMed Scopus (539) Google Scholar). Thus, it is reasonable to hypothesize that FasL expressed on the endothelial surface may induce apoptosis in Fas-bearing neutrophils, thereby preventing their transmigration to myocardial tissue. In support of this hypothesis, we have shown that neutrophil depletion will abrogate the protective action of the FasL transgene on myocardial ischemia-reperfusion injury. In contrast, expression of membrane-bound FasL is reported to promote neutrophil chemotaxis (42Shudo K. Kinoshita K. Imamura R. Fan H. Hasumoto K. Tanaka M. Nagata S. Suda T. Eur. J. Immunol. 2001; 31: 2504-2511Crossref PubMed Scopus (72) Google Scholar, 43Gregory M.S. Repp A.C. Holhbaum A.M. Saff R.R. Marshak-Rothstein A. Ksander B.R. J. Immunol. 2002; 169: 2727-2735Crossref PubMed Scopus (72) Google Scholar, 44Sano Y. Yamada J. Ishino Y. Adachi W. Kawasaki S. Suzuki T. Kinoshita S. Okuyama T. Azuma N. Exp. Eye Res. 2002; 75: 475-483Crossref PubMed Google Scholar), although FasL has no direct chemoattractant activity (45Behrens C.K. Igney F.H. Arnold B. Moller P. Krammer P.H. J. Immunol. 2001; 166: 3240-3247Crossref PubMed Scopus (33) Google Scholar). In these situations, neutrophil chemotaxis may be the indirect result of pro-inflammatory cytokine release that is caused by the cytotoxic activity of FasL (12Miwa K. Asano M. Horai R. Iwakura Y. Nagata S. Suda T. Nat. Med. 1998; 4: 1287-1292Crossref PubMed Scopus (344) Google Scholar,13Hohlbaum A.M. Gregory M.S. Ju S.T. Marshak-Rothstein A. J. Immunol. 2001; 167: 6217-6224Crossref PubMed Scopus (136) Google Scholar). These effects may be minimized in the VEcadFasL mice due to the resistance of endothelial cells to Fas-mediated apoptosis (4Sata M. Suhara T. Walsh K. Arterioscler. Thromb. Vasc. Biol. 2000; 20: 309-316Crossref PubMed Scopus (111) Google Scholar, 5Suhara T. Mano T. Oliveira B.E. Walsh K. Circ. Res. 2001; 89: 13-19Crossref PubMed Scopus (164) Google Scholar, 46Sata M. Nishimatsu H. Suzuki E. Sugiura S. Yoshizumi M. Ouchi Y. Hirata Y. Nagai R. FASEB J. 2001; 15: 2530-2532Crossref PubMed Scopus (110) Google Scholar) and to the co-expression of immunosuppressive cytokines by vascular cells (47Chen J.-J. Sun Y. Nabel G.J. Science. 1998; 282: 1714-1717Crossref PubMed Scopus (336) Google Scholar, 48Nikol S. Isner J.M. Pickering J.G. Kearney M. Leclerc G. Weir L. J. Clin. Invest. 1992; 90: 1582-1592Crossref PubMed Scopus (351) Google Scholar), potentially creating a microenvironment that promotes immunological tolerance to FasL.In summary, we constructed strains of mice that overexpress FasL on the vascular endothelium. These mice were protected from ischemia-reperfusion injury in the heart, displaying smaller infarct size, improved performance, and diminished neutrophil infiltration. These data show that endothelial cells can be engineered to express high levels of FasL, leading to reductions in injury-induced inflammation and organ damage. Fas ligand (FasL)1 is a type II membrane protein belonging to the tumor necrosis factor (TNF) family. It induces apoptosis in various cell types that bear the receptor Fas (also called APO-1 or CD95) (1Nagata S. Golstein P. Science. 1995; 267: 1449-1456Crossref PubMed Scopus (3965) Google Scholar). FasL and Fas-deficient mice, gld and lpr mice, respectively, develop similar phenotypes including splenomegaly, lymphadenopathy, and the accumulation of autoantibodies (2Cohen P.L. Eisenberg R.A. Annu. Rev. Immunol. 1991; 9: 243-269Crossref PubMed Scopus (1131) Google Scholar). These phenotypes suggest that this receptor/ligand pair functions to regulate immune cell homeostasis. Cells differ markedly in their sensitivity to Fas-mediated apoptosis due in part to differences in the expression of FLIP, an inhibitor of Fas-mediated death signaling (3Tschopp J. Irmler M. Thome M. Curr. Opin. Immunol. 1998; 10: 552-558Crossref PubMed Scopus (467) Google Scholar). Endothelial cells express high levels of FLIP and are relatively resistant to FasL-induced apoptosis (4Sata M. Suhara T. Walsh K. Arterioscler. Thromb. Vasc. Biol. 2000; 20: 309-316Crossref PubMed Scopus (111) Google Scholar, 5Suhara T. Mano T. Oliveira B.E. Walsh K. Circ. Res. 2001; 89: 13-19Crossref PubMed Scopus (164) Google Scholar, 6Sata M. Walsh K. J. Biol. Chem. 1998; 273: 33103-33106Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar). In contrast, cardiac myocytes will undergo apoptotic cell death when presented with membrane-bound FasL (7Lee P. Sata M. Lefer D.J. Factor S.M. Walsh K. Kitsis R.N. Am. J. Physiol. 2003; 284: H456-H463Crossref PubMed Scopus (29) Google Scholar). Furthermore, FLIP levels are down-regulated in ischemic regions of the heart following reperfusion (8Rasper D.M. Vaillancourt J.P. Hadano S. Houtzager V.M. Seiden I. Keen S.L.C. Tawa P. Xanthoudakis S. Nasir J. Martindale D. Koop B.F. Peterson E.P. Thornberry N.A. Huang J.Q. Macpherson D.P. Black S.C. Hornung F. Lenardo M.J. Hayden M.R. Roy S. Nicholson D.W. Cell Death Differ. 1998; 5: 271-288Crossref PubMed Scopus (279) Google Scholar), suggesting that Fas-mediated apoptosis may contribute to tissue damage under these conditions. The role of FasL in inflammation has received considerable attention. FasL expression is detected at some immune privileged sites, for example, eye and testis (9Griffith T.S. Brunner T. Fletcher S.M. Green D.R. Ferguson T.A. Science. 1995; 270: 1189-1192Crossref PubMed Scopus (1863) Google Scholar, 10Bellgrau D. Gold D. Selawry H. Moore J. Franzusoff A. Duke R.C. Nature. 1995; 377: 630-632Crossref PubMed Scopus (1097) Google Scholar), where it is believed to protect those tissues by inducing apoptosis in the intruding Fas-bearing immune cells (9Griffith T.S. Brunner T. Fletcher S.M. Green D.R. Ferguson T.A. Science. 1995; 270: 1189-1192Crossref PubMed Scopus (1863) Google Scholar). Although some studies have shown that FasL expression can promote allograft survival in transplantation models, other studies have reported that ectopic FasL promotes neutrophil infiltration and accelerates rejection (reviewed in Ref. 11Green D.R. Ferguson T.A. Nat. Rev. Mol. Cell. Biol. 2001; 2: 917-924Crossref PubMed Scopus (214) Google Scholar). FasL-induced inflammatory responses can result from the release of interleukin-1β (12Miwa K. Asano M. Horai R. Iwakura Y. Nagata S. Suda T. Nat. Med. 1998; 4: 1287-1292Crossref PubMed Scopus (344) Google Scholar) and other cytokines that recruit neutrophils (13Hohlbaum A.M. Gregory M.S. Ju S.T. Marshak-Rothstein A. J. Immunol. 2001; 167: 6217-6224Crossref PubMed Scopus (136) Google Scholar). In this regard, it has been shown that ectopic FasL expression in cardiac myocytes promotes proinflammatory cytokine expression, leukocyte infiltration, and hypertrophy (14Nelson D.P. Setser E. Hall D.G. Schwartz S.M. Hewitt T. Klevitsky R. Osinska H. Bellgrau D. Duke R.C. Robbins J. J. Clin. Invest. 2000; 105: 1199-1208Crossref PubMed Scopus (75) Google Scholar). Heart grafts overexpressing FasL in cardiac myocytes also undergo accelerated rejection, accompanied by massive neutrophil infiltration (15Takeuchi T. Ueki T. Nishimatsu H. Kajiwara T. Ishida T. Jishage K. Ueda O. Suzuki H. Li B. Moriyama N. Kitamura T. J. Immunol. 1999; 162: 518-522PubMed Google Scholar). Inflammatory reactions involving leukocytes have been shown to contribute to the pathogenesis of myocardial ischemia-reperfusion injury (MI/R) (16Frangogiannis N.G. Smith C.W. Entman M.L. Cardiovasc. Res. 2002; 53: 31-47Crossref PubMed Scopus (1704) Google Scholar). Necrotic cardiomyocytes release cytokines causing leukocytes to transmigrate through the endothelium and accumulate at the ischemic site. Once in the myocardium, leukocytes release proteolytic enzymes and reactive oxygen species, causing further damage to the heart. Neutrophils play a critical role in reperfusion injury, and it has been shown that neutrophil depletion results in reduced myocardial infarct size (17Litt M.R. Jeremy R.W. Weisman H.F. Winkelstein J.A. Becker L.C. Circulation. 1989; 80: 1816-1827Crossref PubMed Scopus (301) Google Scholar, 18Romson J.L. Hook B.G. Kunkel S.L. Abrams G.D. Schork M.A. Lucchesi B.R. Circulation. 1983; 67: 1016-1023Crossref PubMed Scopus (1028) Google Scholar). Besides its expression on immune cells, FasL is also expressed at low levels on the vascular endothelium (4Sata M. Suhara T. Walsh K. Arterioscler. Thromb. Vasc. Biol. 2000; 20: 309-316Crossref PubMed Scopus (111) Google Scholar). Administration of the pro-inflammatory cytokine TNF-α down-regulates FasL expression in endothelial cells, and overexpression of FasL on the endothelium attenuates leukocyte extravasation induced by TNF-α (19Sata M. Walsh K. Nat. Med. 1998; 4: 415-420Crossref PubMed Scopus (208) Google Scholar). It has also been shown that FasL overexpression on the vascular endothelium of carotid arteries inhibits transplant-associated intimal hyperplasia (20Sata M. Luo Z. Walsh K. J. Immunol. 2001; 166: 6964-6971Crossref PubMed Scopus (50) Google Scholar) and that adenovirus-mediated delivery of FasL to injured vessels inhibits intimal hyperplasia in animals with preexisting immunity to adenovirus (21Luo Z. Sata M. Nguyen T. Kaplan J.M. Akita G.Y. Walsh K. Circulation. 1999; 99: 1776-1779Crossref PubMed Scopus (79) Google Scholar, 22Sata M. Perlman H. Muruve D.A. Silver M. Ikebe M. Libermann T.A. Oettgen P. Walsh K. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 1213-1217Crossref PubMed Scopus (135) Google Scholar). These data suggest that when expressed in endothelial cells, FasL may have anti-inflammatory actions. To elucidate further the function of FasL in endothelium, we constructed transgenic mouse lines that stably express FasL in vascular endothelium under the control of the vascular endothelial cadherin (VEcadherin) promoter. When subjected to ischemia-reperfusion, the hearts of VEcadFasL transgenic mice displayed significantly smaller infarct size and a reduction in functional impairment when compared with nontransgenic mice. Consistent with these data, a decrease in neutrophil activity was observed at an early time point in transgenic hearts compared with nontransgenic hearts. No differences in infarct size were seen between transgenic and nontransgenic mice that were treated with cytotoxic anti-neutrophil serum prior to injury. These data support the hypothesis that endothelial FasL acts to minimize neutrophil extravasation and organ damage following ischemia-reperfusion injury. DISCUSSIONIn this study we described the effects of endothelial FasL overexpression on myocardial damage following ischemia-reperfusion injury. We established a new mouse model that specifically expresses FasL in vascular endothelial cells. These mice developed normally and had normal hearts but exhibited a 42% reduction in infarct size following myocardial ischemia-reperfusion injury. Consistent with the smaller infarct size, VEcadFasL mouse hearts displayed less functional impairment than nontransgenic mice following the injury. The cardioprotective effect in the transgenic mice may be attributable to a 54% reduction in neutrophil accumulation in the reperfused tissue. Thus, overexpression of FasL on vascular endothelium may serve to minimize neutrophil recruitment, thereby reducing myocardial damage.A number of studies have analyzed the role of the Fas/FasL system in the heart. The α-MyHC-FasL transgenic mice that ectopically express FasL in cardiac myocytes develop mild cardiac hypertrophy associated with interstitial fibrosis and leukocyte inflammation (14Nelson D.P. Setser E. Hall D.G. Schwartz S.M. Hewitt T. Klevitsky R. Osinska H. Bellgrau D. Duke R.C. Robbins J. J. Clin. Invest. 2000; 105: 1199-1208Crossref PubMed Scopus (75) Google Scholar). Although apoptosis was not observed in the α-MyHC-FasL hearts, other experiments have provided direct evidence that Fas pathway can induce apoptosis in cardiomyocytes. In the study of Lee et al. (7Lee P. Sata M. Lefer D.J. Factor S.M. Walsh K. Kitsis R.N. Am. J. Physiol. 2003; 284: H456-H463Crossref PubMed Scopus (29) Google Scholar), it was shown that FasL expression promotes cardiomyocyte apoptosis bothin vivo and in vitro and that Fas-deficientlpr mice displayed less cardiomyocyte apoptosis and correspondingly smaller infarct size after ischemia-reperfusion injury. Hearts from lpr mice also display fewer apoptotic cellsex vivo after ischemia-reperfusion (31Jeremias I. Kupatt C. Martin-Villalba A. Habazettl H. Schenkel J. Boekstegers P. Debatin K.M. Circulation. 2000; 102: 915-920Crossref PubMed Scopus (192) Google Scholar), and intravenous administration of FasL-blocking antibody attenuates inflammation in the heart and reduces myocardial infarct size following ischemia-reperfusion injury (32Shiraishi H. Toyozaki T. Tsukamoto Y. Saito T. Masuda Y. Hiroshima K. Ohwada H. Kobayashi N. Hiroe M. Lab. Invest. 2002; 82: 1121-1129Crossref PubMed Scopus (13) Google Scholar). It is possible that the inflammation and hypertrophy in α-MyHC-FasL mouse hearts result from chronic, but undetectably low, levels of apoptosis in cardiac myocytes or tissue-resident inflammatory cells (13Hohlbaum A.M. Gregory M.S. Ju S.T. Marshak-Rothstein A. J. Immunol. 2001; 167: 6217-6224Crossref PubMed Scopus (136) Google Scholar). Alternatively, the cardiac hypertrophy observed in α-MyHC-FasL mice may result from an activation of intrinsic growth regulatory signaling cascades that promote the phosphorylation of glycogen synthase kinase 3β within cardiac myocytes (33Badorff C. Ruetten H. Mueller S. Stahmer M. Gehring D. Jung F. Ihling C. Zeiher A.M. Dimmeler S. J. Clin. Invest. 2002; 109: 373-381Crossref PubMed Scopus (145) Google Scholar). In this regard, Fas is reported to positively affect the growth of a number of cell types (reviewed in Ref. 34Budd R.C. J. Clin. Invest. 2002; 109: 437-441Crossref PubMed Scopus (131) Google Scholar); however, cell cycle entry in some of these cases may result from the execution of the apoptotic process (for example see Ref. 35Perlman H. Sata M. Krasinski K. Dorai T. Buttyan R. Walsh K. Cardiovasc. Res. 2000; 45: 570-578Crossref PubMed Scopus (42) Google Scholar).In contrast to cardiomyocytes, vascular endothelial cells express FasL, and overexpression of FasL in these cells normally does not affect viability or cell cycle status (4Sata M. Suhara T. Walsh K. Arterioscler. Thromb. Vasc. Biol. 2000; 20: 309-316Crossref PubMed Scopus (111) Google Scholar). Thus, the overexpression of FasL by the endothelial cells of transgenic mice would be expected to alter immune responses in the absence of cytotoxic or proliferative effects on the endothelium. The marked differences between the heart phenotypes of VEcadFasL and α-MyHC-FasL mice clearly illustrate that FasL can exert either pro-inflammatory or anti-inflammatory actions within an organ depending upon differences in cell type-specific expression or microenvironment. Ectopic expression of FasL in cardiac myocytes leads to inflammation and tissue damage, whereas overexpression of FasL on the vascular endothelium has an anti-inflammatory effect and protects the heart from injury. Similar to our findings in heart, both pro- and anti-inflammatory effects of FasL expression have also been observed in other transgenic models. Transgenic mice expressing FasL in pancreatic beta cells display neutrophil infiltration and develop diabetes at a young age (36Kang S.M. Schneider D.B. Lin Z. Hanahan D. Dichek D.A. Stock P.G. Baekkeskov S. Nat. Med. 1997; 3: 738-743Crossref PubMed Scopus (428) Google Scholar). In this setting, ectopic expression of FasL will lead to the destruction of Fas-positive islet cells (37Muruve D.A. Nicolson A.G. Manfro R.C. Strom T.B. Sukhatme V.P. Libermann T.A. Hum. Gene Ther. 1997; 8: 955-963Crossref PubMed Scopus (69) Google Scholar). In contrast, FasL is expressed on follicular cells of the thyroid (38Stassi G. Di Liberto D. Todaro M. Zeuner A. Ricci-Vitiani L. Stoppacciaro A. Ruco L. Farina F. Zummo G. De Maria R. Nat. Immunol. 2000; 1: 483-488Crossref PubMed Scopus (133) Google Scholar), and transgenic expression of FasL on thyroid follicular cells prevents thyroid allograft rejection (39Tourneur L. Malassagne B. Batteux F. Fabre M. Mistou S. Lallemand E. Lores P. Chiocchia G. J. Immunol. 2001; 167: 1338-1346Crossref PubMed Scopus (31) Google Scholar). These data support the hypothesis that FasL-induced inflammation involves FasL that is expressed ectopically, whereas anti-inflammatory effects are associated with overexpression in cell types that normally express endogenous FasL (11Green D.R. Ferguson T.A. Nat. Rev. Mol. Cell. Biol. 2001; 2: 917-924Crossref PubMed Scopus (214) Google Scholar).Neutrophils accumulate at the site of ischemia within hours of reperfusion and damage the tissue by releasing proteases and oxidants (40Dreyer W.J. Michael L.H. West M.S. Smith C.W. Rothlein R. Rossen R.D. Anderson D.C. Entman M.L. Circulation. 1991; 84: 400-411Crossref PubMed Scopus (258) Google Scholar). It is well established that neutrophil depletion will reduce ischemia-reperfusion injury in the heart (17Litt M.R. Jeremy R.W. Weisman H.F. Winkelstein J.A. Becker L.C. Circulation. 1989; 80: 1816-1827Crossref PubMed Scopus (301) Google Scholar, 18Romson J.L. Hook B.G. Kunkel S.L. Abrams G.D. Schork M.A. Lucchesi B.R. Circulation. 1983; 67: 1016-1023Crossref PubMed Scopus (1028) Google Scholar). Neutrophils express both Fas and FasL and are susceptible to Fas-mediated cell death (41Liles W.C. Kiener P.A. Ledbetter J.A. Aruffo A. Klebanoff S.J. J. Exp. Med. 1996; 184: 429-440Crossref PubMed Scopus (539) Google Scholar). Thus, it is reasonable to hypothesize that FasL expressed on the endothelial surface may induce apoptosis in Fas-bearing neutrophils, thereby preventing their transmigration to myocardial tissue. In support of this hypothesis, we have shown that neutrophil depletion will abrogate the protective action of the FasL transgene on myocardial ischemia-reperfusion injury. In contrast, expression of membrane-bound FasL is reported to promote neutrophil chemotaxis (42Shudo K. Kinoshita K. Imamura R. Fan H. Hasumoto K. Tanaka M. Nagata S. Suda T. Eur. J. Immunol. 2001; 31: 2504-2511Crossref PubMed Scopus (72) Google Scholar, 43Gregory M.S. Repp A.C. Holhbaum A.M. Saff R.R. Marshak-Rothstein A. Ksander B.R. J. Immunol. 2002; 169: 2727-2735Crossref PubMed Scopus (72) Google Scholar, 44Sano Y. Yamada J. Ishino Y. Adachi W. Kawasaki S. Suzuki T. Kinoshita S. Okuyama T. Azuma N. Exp. Eye Res. 2002; 75: 475-483Crossref PubMed Google Scholar), although FasL has no direct chemoattractant activity (45Behrens C.K. Igney F.H. Arnold B. Moller P. Krammer P.H. J. Immunol. 2001; 166: 3240-3247Crossref PubMed Scopus (33) Google Scholar). In these situations, neutrophil chemotaxis may be the indirect result of pro-inflammatory cytokine release that is caused by the cytotoxic activity of FasL (12Miwa K. Asano M. Horai R. Iwakura Y. Nagata S. Suda T. Nat. Med. 1998; 4: 1287-1292Crossref PubMed Scopus (344) Google Scholar,13Hohlbaum A.M. Gregory M.S. Ju S.T. Marshak-Rothstein A. J. Immunol. 2001; 167: 6217-6224Crossref PubMed Scopus (136) Google Scholar). These effects may be minimized in the VEcadFasL mice due to the resistance of endothelial cells to Fas-mediated apoptosis (4Sata M. Suhara T. Walsh K. Arterioscler. Thromb. Vasc. Biol. 2000; 20: 309-316Crossref PubMed Scopus (111) Google Scholar, 5Suhara T. Mano T. Oliveira B.E. Walsh K. Circ. Res. 2001; 89: 13-19Crossref PubMed Scopus (164) Google Scholar, 46Sata M. Nishimatsu H. Suzuki E. Sugiura S. Yoshizumi M. Ouchi Y. Hirata Y. Nagai R. FASEB J. 2001; 15: 2530-2532Crossref PubMed Scopus (110) Google Scholar) and to the co-expression of immunosuppressive cytokines by vascular cells (47Chen J.-J. Sun Y. Nabel G.J. Science. 1998; 282: 1714-1717Crossref PubMed Scopus (336) Google Scholar, 48Nikol S. Isner J.M. Pickering J.G. Kearney M. Leclerc G. Weir L. J. Clin. Invest. 1992; 90: 1582-1592Crossref PubMed Scopus (351) Google Scholar), potentially creating a microenvironment that promotes immunological tolerance to FasL.In summary, we constructed strains of mice that overexpress FasL on the vascular endothelium. These mice were protected from ischemia-reperfusion injury in the heart, displaying smaller infarct size, improved performance, and diminished neutrophil infiltration. These data show that endothelial cells can be engineered to express high levels of FasL, leading to reductions in injury-induced inflammation and organ damage. In this study we described the effects of endothelial FasL overexpression on myocardial damage following ischemia-reperfusion injury. We established a new mouse model that specifically expresses FasL in vascular endothelial cells. These mice developed normally and had normal hearts but exhibited a 42% reduction in infarct size following myocardial ischemia-reperfusion injury. Consistent with the smaller infarct size, VEcadFasL mouse hearts displayed less functional impairment than nontransgenic mice following the injury. The cardioprotective effect in the transgenic mice may be attributable to a 54% reduction in neutrophil accumulation in the reperfused tissue. Thus, overexpression of FasL on vascular endothelium may serve to minimize neutrophil recruitment, thereby reducing myocardial damage. A number of studies have analyzed the role of the Fas/FasL system in the heart. The α-MyHC-FasL transgenic mice that ectopically express FasL in cardiac myocytes develop mild cardiac hypertrophy associated with interstitial fibrosis and leukocyte inflammation (14Nelson D.P. Setser E. Hall D.G. Schwartz S.M. Hewitt T. Klevitsky R. Osinska H. Bellgrau D. Duke R.C. Robbins J. J. Clin. Invest. 2000; 105: 1199-1208Crossref PubMed Scopus (75) Google Scholar). Although apoptosis was not observed in the α-MyHC-FasL hearts, other experiments have provided direct evidence that Fas pathway can induce apoptosis in cardiomyocytes. In the study of Lee et al. (7Lee P. Sata M. Lefer D.J. Factor S.M. Walsh K. Kitsis R.N. Am. J. Physiol. 2003; 284: H456-H463Crossref PubMed Scopus (29) Google Scholar), it was shown that FasL expression promotes cardiomyocyte apoptosis bothin vivo and in vitro and that Fas-deficientlpr mice displayed less cardiomyocyte apoptosis and correspondingly smaller infarct size after ischemia-reperfusion injury. Hearts from lpr mice also display fewer apoptotic cellsex vivo after ischemia-reperfusion (31Jeremias I. Kupatt C. Martin-Villalba A. Habazettl H. Schenkel J. Boekstegers P. Debatin K.M. Circulation. 2000; 102: 915-920Crossref PubMed Scopus (192) Google Scholar), and intravenous administration of FasL-blocking antibody attenuates inflammation in the heart and reduces myocardial infarct size following ischemia-reperfusion injury (32Shiraishi H. Toyozaki T. Tsukamoto Y. Saito T. Masuda Y. Hiroshima K. Ohwada H. Kobayashi N. Hiroe M. Lab. Invest. 2002; 82: 1121-1129Crossref PubMed Scopus (13) Google Scholar). It is possible that the inflammation and hypertrophy in α-MyHC-FasL mouse hearts result from chronic, but undetectably low, levels of apoptosis in cardiac myocytes or tissue-resident inflammatory cells (13Hohlbaum A.M. Gregory M.S. Ju S.T. Marshak-Rothstein A. J. Immunol. 2001; 167: 6217-6224Crossref PubMed Scopus (136) Google Scholar). Alternatively, the cardiac hypertrophy observed in α-MyHC-FasL mice may result from an activation of intrinsic growth regulatory signaling cascades that promote the phosphorylation of glycogen synthase kinase 3β within cardiac myocytes (33Badorff C. Ruetten H. Mueller S. Stahmer M. Gehring D. Jung F. Ihling C. Zeiher A.M. Dimmeler S. J. Clin. Invest. 2002; 109: 373-381Crossref PubMed Scopus (145) Google Scholar). In this regard, Fas is reported to positively affect the growth of a number of cell types (reviewed in Ref. 34Budd R.C. J. Clin. Invest. 2002; 109: 437-441Crossref PubMed Scopus (131) Google Scholar); however, cell cycle entry in some of these cases may result from the execution of the apoptotic process (for example see Ref. 35Perlman H. Sata M. Krasinski K. Dorai T. Buttyan R. Walsh K. Cardiovasc. Res. 2000; 45: 570-578Crossref PubMed Scopus (42) Google Scholar). In contrast to cardiomyocytes, vascular endothelial cells express FasL, and overexpression of FasL in these cells normally does not affect viability or cell cycle status (4Sata M. Suhara T. Walsh K. Arterioscler. Thromb. Vasc. Biol. 2000; 20: 309-316Crossref PubMed Scopus (111) Google Scholar). Thus, the overexpression of FasL by the endothelial cells of transgenic mice would be expected to alter immune responses in the absence of cytotoxic or proliferative effects on the endothelium. The marked differences between the heart phenotypes of VEcadFasL and α-MyHC-FasL mice clearly illustrate that FasL can exert either pro-inflammatory or anti-inflammatory actions within an organ depending upon differences in cell type-specific expression or microenvironment. Ectopic expression of FasL in cardiac myocytes leads to inflammation and tissue damage, whereas overexpression of FasL on the vascular endothelium has an anti-inflammatory effect and protects the heart from injury. Similar to our findings in heart, both pro- and anti-inflammatory effects of FasL expression have also been observed in other transgenic models. Transgenic mice expressing FasL in pancreatic beta cells display neutrophil infiltration and develop diabetes at a young age (36Kang S.M. Schneider D.B. Lin Z. Hanahan D. Dichek D.A. Stock P.G. Baekkeskov S. Nat. Med. 1997; 3: 738-743Crossref PubMed Scopus (428) Google Scholar). In this setting, ectopic expression of FasL will lead to the destruction of Fas-positive islet cells (37Muruve D.A. Nicolson A.G. Manfro R.C. Strom T.B. Sukhatme V.P. Libermann T.A. Hum. Gene Ther. 1997; 8: 955-963Crossref PubMed Scopus (69) Google Scholar). In contrast, FasL is expressed on follicular cells of the thyroid (38Stassi G. Di Liberto D. Todaro M. Zeuner A. Ricci-Vitiani L. Stoppacciaro A. Ruco L. Farina F. Zummo G. De Maria R. Nat. Immunol. 2000; 1: 483-488Crossref PubMed Scopus (133) Google Scholar), and transgenic expression of FasL on thyroid follicular cells prevents thyroid allograft rejection (39Tourneur L. Malassagne B. Batteux F. Fabre M. Mistou S. Lallemand E. Lores P. Chiocchia G. J. Immunol. 2001; 167: 1338-1346Crossref PubMed Scopus (31) Google Scholar). These data support the hypothesis that FasL-induced inflammation involves FasL that is expressed ectopically, whereas anti-inflammatory effects are associated with overexpression in cell types that normally express endogenous FasL (11Green D.R. Ferguson T.A. Nat. Rev. Mol. Cell. Biol. 2001; 2: 917-924Crossref PubMed Scopus (214) Google Scholar). Neutrophils accumulate at the site of ischemia within hours of reperfusion and damage the tissue by releasing proteases and oxidants (40Dreyer W.J. Michael L.H. West M.S. Smith C.W. Rothlein R. Rossen R.D. Anderson D.C. Entman M.L. Circulation. 1991; 84: 400-411Crossref PubMed Scopus (258) Google Scholar). It is well established that neutrophil depletion will reduce ischemia-reperfusion injury in the heart (17Litt M.R. Jeremy R.W. Weisman H.F. Winkelstein J.A. Becker L.C. Circulation. 1989; 80: 1816-1827Crossref PubMed Scopus (301) Google Scholar, 18Romson J.L. Hook B.G. Kunkel S.L. Abrams G.D. Schork M.A. Lucchesi B.R. Circulation. 1983; 67: 1016-1023Crossref PubMed Scopus (1028) Google Scholar). Neutrophils express both Fas and FasL and are susceptible to Fas-mediated cell death (41Liles W.C. Kiener P.A. Ledbetter J.A. Aruffo A. Klebanoff S.J. J. Exp. Med. 1996; 184: 429-440Crossref PubMed Scopus (539) Google Scholar). Thus, it is reasonable to hypothesize that FasL expressed on the endothelial surface may induce apoptosis in Fas-bearing neutrophils, thereby preventing their transmigration to myocardial tissue. In support of this hypothesis, we have shown that neutrophil depletion will abrogate the protective action of the FasL transgene on myocardial ischemia-reperfusion injury. In contrast, expression of membrane-bound FasL is reported to promote neutrophil chemotaxis (42Shudo K. Kinoshita K. Imamura R. Fan H. Hasumoto K. Tanaka M. Nagata S. Suda T. Eur. J. Immunol. 2001; 31: 2504-2511Crossref PubMed Scopus (72) Google Scholar, 43Gregory M.S. Repp A.C. Holhbaum A.M. Saff R.R. Marshak-Rothstein A. Ksander B.R. J. Immunol. 2002; 169: 2727-2735Crossref PubMed Scopus (72) Google Scholar, 44Sano Y. Yamada J. Ishino Y. Adachi W. Kawasaki S. Suzuki T. Kinoshita S. Okuyama T. Azuma N. Exp. Eye Res. 2002; 75: 475-483Crossref PubMed Google Scholar), although FasL has no direct chemoattractant activity (45Behrens C.K. Igney F.H. Arnold B. Moller P. Krammer P.H. J. Immunol. 2001; 166: 3240-3247Crossref PubMed Scopus (33) Google Scholar). In these situations, neutrophil chemotaxis may be the indirect result of pro-inflammatory cytokine release that is caused by the cytotoxic activity of FasL (12Miwa K. Asano M. Horai R. Iwakura Y. Nagata S. Suda T. Nat. Med. 1998; 4: 1287-1292Crossref PubMed Scopus (344) Google Scholar,13Hohlbaum A.M. Gregory M.S. Ju S.T. Marshak-Rothstein A. J. Immunol. 2001; 167: 6217-6224Crossref PubMed Scopus (136) Google Scholar). These effects may be minimized in the VEcadFasL mice due to the resistance of endothelial cells to Fas-mediated apoptosis (4Sata M. Suhara T. Walsh K. Arterioscler. Thromb. Vasc. Biol. 2000; 20: 309-316Crossref PubMed Scopus (111) Google Scholar, 5Suhara T. Mano T. Oliveira B.E. Walsh K. Circ. Res. 2001; 89: 13-19Crossref PubMed Scopus (164) Google Scholar, 46Sata M. Nishimatsu H. Suzuki E. Sugiura S. Yoshizumi M. Ouchi Y. Hirata Y. Nagai R. FASEB J. 2001; 15: 2530-2532Crossref PubMed Scopus (110) Google Scholar) and to the co-expression of immunosuppressive cytokines by vascular cells (47Chen J.-J. Sun Y. Nabel G.J. Science. 1998; 282: 1714-1717Crossref PubMed Scopus (336) Google Scholar, 48Nikol S. Isner J.M. Pickering J.G. Kearney M. Leclerc G. Weir L. J. Clin. Invest. 1992; 90: 1582-1592Crossref PubMed Scopus (351) Google Scholar), potentially creating a microenvironment that promotes immunological tolerance to FasL. In summary, we constructed strains of mice that overexpress FasL on the vascular endothelium. These mice were protected from ischemia-reperfusion injury in the heart, displaying smaller infarct size, improved performance, and diminished neutrophil infiltration. These data show that endothelial cells can be engineered to express high levels of FasL, leading to reductions in injury-induced inflammation and organ damage. We thank A. Hofmann for providing the pBSmVEhFasL plasmid.
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