A Constitutive Cytoprotective Pathway Protects Endothelial Cells from Lipopolysaccharide-induced Apoptosis
2001; Elsevier BV; Volume: 276; Issue: 18 Linguagem: Inglês
10.1074/jbc.m100819200
ISSN1083-351X
AutoresDouglas D. Bannerman, Joan C. Tupper, William A. Ricketts, C. Frank Bennett, Robert K. Winn, John M. Harlan,
Tópico(s)Neutrophil, Myeloperoxidase and Oxidative Mechanisms
ResumoLipopolysaccharide (LPS) has been implicated as the bacterial component responsible for much of the endothelial cell injury/dysfunction associated with Gram-negative bacterial infections. Protein synthesis inhibition is required to sensitize the endothelium to lipopolysaccharide-induced apoptosis, suggesting that a constitutive or inducible cytoprotective protein(s) is required for endothelial survival. We have identified two known endothelial anti-apoptotic proteins, c-FLIP and Mcl-1, the expression of which is decreased markedly in the presence of cycloheximide. Decreased expression of both proteins preceded apoptosis evoked by lipopolysaccharide + cycloheximide. Caspase inhibition protected against apoptosis, but not the decreased expression of c-FLIP and Mcl-1, suggesting that they exert protection upstream of caspase activation. Inhibition of the degradation of these two proteins with the proteasome inhibitor, lactacystin, prevented lipopolysaccharide + cycloheximide-induced apoptosis. Similarly, lactacystin protected against endothelial apoptosis induced by either tumor necrosis factor-α or interleukin-1β in the presence of cycloheximide. That apoptosis could be blocked in the absence of new protein synthesis by inhibition of the proteasome degradative pathway implicates the requisite involvement of a constitutively expressed protein(s) in the endothelial cytoprotective pathway. Finally, reduction of FLIP expression with antisense oligonucleotides sensitized endothelial cells to LPS killing, demonstrating a definitive role for FLIP in the protection of endothelial cells from LPS-induced apoptosis. Lipopolysaccharide (LPS) has been implicated as the bacterial component responsible for much of the endothelial cell injury/dysfunction associated with Gram-negative bacterial infections. Protein synthesis inhibition is required to sensitize the endothelium to lipopolysaccharide-induced apoptosis, suggesting that a constitutive or inducible cytoprotective protein(s) is required for endothelial survival. We have identified two known endothelial anti-apoptotic proteins, c-FLIP and Mcl-1, the expression of which is decreased markedly in the presence of cycloheximide. Decreased expression of both proteins preceded apoptosis evoked by lipopolysaccharide + cycloheximide. Caspase inhibition protected against apoptosis, but not the decreased expression of c-FLIP and Mcl-1, suggesting that they exert protection upstream of caspase activation. Inhibition of the degradation of these two proteins with the proteasome inhibitor, lactacystin, prevented lipopolysaccharide + cycloheximide-induced apoptosis. Similarly, lactacystin protected against endothelial apoptosis induced by either tumor necrosis factor-α or interleukin-1β in the presence of cycloheximide. That apoptosis could be blocked in the absence of new protein synthesis by inhibition of the proteasome degradative pathway implicates the requisite involvement of a constitutively expressed protein(s) in the endothelial cytoprotective pathway. Finally, reduction of FLIP expression with antisense oligonucleotides sensitized endothelial cells to LPS killing, demonstrating a definitive role for FLIP in the protection of endothelial cells from LPS-induced apoptosis. endothelial cell lipopolysaccharide tumor necrosis factor-α interleukin Toll-like receptor, CHX, cycloheximide z-VAD-fluoromethylketone phosphate-buffered saline polyvinylidene fluoride membrane horseradish peroxidase immunoglobulin G poly(ADP-ribose) polymerase death domain cellular inhibitor of apoptosis enzyme-linked immunosorbent assay vascular cell adhesion molecule-1 TNF receptor-associated death domain Fas-associated death domain Despite advances in anti-microbial therapy and overall medical care, Gram-negative bacterial sepsis remains a common, life-threatening event. The challenge of managing septic patients is compounded by the development of key vascular complications including, systemic vascular collapse, disseminated intravascular coagulation, and vascular leak syndromes (1Brigham K.L. Meyrick B. Am. Rev. Respir. Dis. 1986; 133: 913-927PubMed Google Scholar, 2Hinshaw L.B. Crit. Care Med. 1996; 24: 1072-1078Crossref PubMed Scopus (309) Google Scholar, 3Levi M. ten Cate H. van der Poll T. van Deventer S.J. JAMA. 1993; 270: 975-979Crossref PubMed Scopus (342) Google Scholar, 4Thijs L.G. de Boer J.P. de Groot M.C. Hack C.E. Intensive Care Med. 1993; 19: S8-15Crossref PubMed Scopus (87) Google Scholar, 5Ulevitch R.J. Cochrane C.G. Henson P.M. Morrison D.C. Doe W.F. J. Exp. Med. 1975; 142: 1570-1590Crossref PubMed Scopus (47) Google Scholar). A common denominator to all these complications is endothelial cell (EC)1 injury and/or dysfunction. Endotoxin or lipopolysaccharide (LPS), which resides in the outer membrane of Gram-negative bacteria, has been implicated as the causative agent responsible for EC dysfunction (1Brigham K.L. Meyrick B. Am. Rev. Respir. Dis. 1986; 133: 913-927PubMed Google Scholar, 5Ulevitch R.J. Cochrane C.G. Henson P.M. Morrison D.C. Doe W.F. J. Exp. Med. 1975; 142: 1570-1590Crossref PubMed Scopus (47) Google Scholar, 6Brandtzaeg P. Kierulf P. Gaustad P. Skulberg A. Bruun J.N. Halvorsen S. Sorensen E. J. Infect. Dis. 1989; 159: 195-204Crossref PubMed Scopus (467) Google Scholar, 7Ziegler E.J. McCutchan J.A. Fierer J. Glauser M.P. Sadoff J.C. Douglas H. Braude A.I. N. Engl. J. Med. 1982; 307: 1225-1230Crossref PubMed Scopus (735) Google Scholar). In the absence of non-endothelial cell-derived host mediator systems, LPS directly evokes numerous EC responses including: 1) up-regulation of adhesion molecules; 2) increased production of cytokines, nitric oxide, and tissue factor; 3) loss of monolayer integrity and barrier function; and 4) apoptosis (8Bannerman D.D. Goldblum S.E. Lab. Invest. 1999; 79: 1181-1199PubMed Google Scholar). In addition to a direct role, LPS stimulates the production of inflammatory cytokines, including interleukin (IL)-1β and tumor necrosis factor (TNF)-α, which also elicit an altered pathophysiological endothelial state (9Thijs L.G. Groeneveld A.B. Hack C.E. Curr. Top. Microbiol. Immunol. 1996; 216: 209-237PubMed Google Scholar).LPS-induced EC apoptosis has been observed both in vitro(10Bannerman D.D. Sathyamoorthy M. Goldblum S.E. J. Biol. Chem. 1998; 273: 35371-35380Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar, 11Choi K.B. Wong F. Harlan J.M. Chaudhary P.M. Hood L. Karsan A. J. Biol. Chem. 1998; 273: 20185-20188Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar, 12Frey E.A. Finlay B.B. Microb. Pathog. 1998; 24: 101-109Crossref PubMed Scopus (71) Google Scholar, 13Hoyt D.G. Mannix R.J. Rusnak J.M. Pitt B.R. Lazo J.S. Am. J. Physiol. 1995; 269: L171-L177PubMed Google Scholar, 14Zen K. Karsan A. Stempien-Otero A. Yee E. Tupper J. Li X. Eunson T. Kay M.A. Wilson C.B. Winn R.K. Harlan J.M. J. Biol. Chem. 1999; 274: 28808-28815Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar) and in vivo (15Fujita M. Kuwano K. Kunitake R. Hagimoto N. Miyazaki H. Kaneko Y. Kawasaki M. Maeyama T. Hara N. Int. Arch. Allergy Immunol. 1998; 117: 202-208Crossref PubMed Scopus (117) Google Scholar, 16Haimovitz-Friedman A. Cordon-Cardo C. Bayoumy S. Garzotto M. McLoughlin M. Gallily R. Edwards III, C.K. Schuchman E.H. Fuks Z. Kolesnick R. J. Exp. Med. 1997; 186: 1831-1841Crossref PubMed Scopus (379) Google Scholar). LPS directly induces apoptosis in bovine and ovine EC (10Bannerman D.D. Sathyamoorthy M. Goldblum S.E. J. Biol. Chem. 1998; 273: 35371-35380Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar, 12Frey E.A. Finlay B.B. Microb. Pathog. 1998; 24: 101-109Crossref PubMed Scopus (71) Google Scholar, 13Hoyt D.G. Mannix R.J. Rusnak J.M. Pitt B.R. Lazo J.S. Am. J. Physiol. 1995; 269: L171-L177PubMed Google Scholar, 17Hoyt D.G. Mannix R.J. Gerritsen M.E. Watkins S.C. Lazo J.S. Pitt B.R. Am. J. Physiol. 1996; 270: L689-L694PubMed Google Scholar). Sensitization of human EC to LPS-induced apoptosis requires the inhibition of either mRNA or protein synthesis (18Pohlman T.H. Harlan J.M. Cell Immunol. 1989; 119: 41-52Crossref PubMed Scopus (87) Google Scholar). This latter finding suggests that either a constitutively expressed protein with a relatively short half-life or an inducible protein is requisite for EC survival following LPS exposure. Similarly, two LPS-inducible cytokines, IL-1β and TNF-α, induce human EC apoptosis only when new gene expression is blocked (18Pohlman T.H. Harlan J.M. Cell Immunol. 1989; 119: 41-52Crossref PubMed Scopus (87) Google Scholar).Apoptosis has been implicated as an important mechanism of in vivo cell death following LPS exposure. Tissues and organs obtained from either patients who have died of sepsis and multi-organ failure (19Hotchkiss R.S. Swanson P.E. Freeman B.D. Tinsley K.W. Cobb J.P. Matuschak G.M. Buchman T.G. Karl I.E. Crit. Care Med. 1999; 27: 1230-1251Crossref PubMed Scopus (1042) Google Scholar) or animal models of endotoxemia and sepsis reveal enhanced apoptotic cell death (16Haimovitz-Friedman A. Cordon-Cardo C. Bayoumy S. Garzotto M. McLoughlin M. Gallily R. Edwards III, C.K. Schuchman E.H. Fuks Z. Kolesnick R. J. Exp. Med. 1997; 186: 1831-1841Crossref PubMed Scopus (379) Google Scholar, 20Hotchkiss R.S. Swanson P.E. Cobb J.P. Jacobson A. Buchman T.G. Karl I.E. Crit. Care Med. 1997; 25: 1298-1307Crossref PubMed Scopus (295) Google Scholar, 21Zhang X.M. Morikawa A. Takahashi K. Jiang G.Z. Kato Y. Sugiyama T. Kawai M. Fukada M. Yokochi T. Microbiol. Immunol. 1994; 38: 669-671Crossref PubMed Scopus (42) Google Scholar). The vascular endothelium is one tissue that is sensitive to LPS-induced apoptosis. In a murine model of sepsis, apoptotic EC have been detected in pulmonary capillaries (20Hotchkiss R.S. Swanson P.E. Cobb J.P. Jacobson A. Buchman T.G. Karl I.E. Crit. Care Med. 1997; 25: 1298-1307Crossref PubMed Scopus (295) Google Scholar). Intravenous administration of endotoxin into rabbits or rats induces EC death and detachment from the artery wall (22Gaynor E. Bouvier C. Spaet T.H. Science. 1970; 170: 986-988Crossref PubMed Scopus (89) Google Scholar, 23Reidy M.A. Schwartz S.M. Lab. Invest. 1983; 48: 25-34PubMed Google Scholar). In mice challenged with either LPS or TNF-α, disseminated EC apoptosis has been reported in the lung, thymus, and intestine (15Fujita M. Kuwano K. Kunitake R. Hagimoto N. Miyazaki H. Kaneko Y. Kawasaki M. Maeyama T. Hara N. Int. Arch. Allergy Immunol. 1998; 117: 202-208Crossref PubMed Scopus (117) Google Scholar, 16Haimovitz-Friedman A. Cordon-Cardo C. Bayoumy S. Garzotto M. McLoughlin M. Gallily R. Edwards III, C.K. Schuchman E.H. Fuks Z. Kolesnick R. J. Exp. Med. 1997; 186: 1831-1841Crossref PubMed Scopus (379) Google Scholar, 24Kawasaki M. Kuwano K. Hagimoto N. Matsuba T. Kunitake R. Tanaka T. Maeyama T. Hara N. Am. J. Pathol. 2000; 157: 597-603Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar). Finally, injection of a broad spectrum caspase inhibitor following LPS administration decreased EC apoptosis in the lung and improved survival in a murine model of acute lung injury (24Kawasaki M. Kuwano K. Hagimoto N. Matsuba T. Kunitake R. Tanaka T. Maeyama T. Hara N. Am. J. Pathol. 2000; 157: 597-603Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar). Together, these in vitro and in vivo studies indicate that the vascular endothelium is a key target of LPS-induced apoptosis.The mechanisms by which LPS activates apoptosis remain unknown. Efforts to elucidate LPS signaling pathways, apoptotic or otherwise, have been hampered by the lack of an identifiable membrane-bound receptor capable of signal transduction. Recently, Toll-like receptor (TLR)-4 has been identified in both cells of monocytic lineage and EC as the receptor responsible for LPS activation of the NF-κB signaling pathway (25Faure E. Equils O. Sieling P.A. Thomas L. Zhang F.X. Kirschning C.J. Polentarutti N. Muzio M. Arditi M. J. Biol. Chem. 2000; 275: 11058-11063Abstract Full Text Full Text PDF PubMed Scopus (506) Google Scholar, 26Hoshino K. Takeuchi O. Kawai T. Sanjo H. Ogawa T. Takeda Y. Takeda K. Akira S. J. Immunol. 1999; 162: 3749-3752PubMed Google Scholar, 27Lien E. Means T.K. Heine H. Yoshimura A. Kusumoto S. Fukase K. Fenton M.J. Oikawa M. Qureshi N. Monks B. Finberg R.W. Ingalls R.R. Golenbock D.T. J. Clin. Invest. 2000; 105: 497-504Crossref PubMed Scopus (683) Google Scholar). Interestingly, MyD88, a TLR-4-binding protein that has a requisite role in the downstream activation of NF-κB in EC, contains a death domain (DD) (28Kopp E.B. Medzhitov R. Curr. Opin. Immunol. 1999; 11: 13-18Crossref PubMed Scopus (586) Google Scholar, 29Zhang F.X. Kirschning C.J. Mancinelli R. Xu X.P. Jin Y. Faure E. Mantovani A. Rothe M. Muzio M. Arditi M. J. Biol. Chem. 1999; 274: 7611-7614Abstract Full Text Full Text PDF PubMed Scopus (535) Google Scholar). DD are conserved regions of amino acids, which facilitate protein-protein interaction. In the well characterized TNF pathway, the TNF receptor-binding protein, TRADD, recruits another adapter protein, FADD, through the interaction of their respective death domains. An additional conserved sequence in FADD, the death effector domain (DED), enables the recruitment of caspase 8, an upstream cysteine protease whose activation initiates a cascade of proteolytic events characteristic of apoptosis. We have previously reported that LPS-induced EC apoptosis is FADD-dependent (11Choi K.B. Wong F. Harlan J.M. Chaudhary P.M. Hood L. Karsan A. J. Biol. Chem. 1998; 273: 20185-20188Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar). Whether FADD is recruited to MyD88 following LPS stimulation via binding of their respective DD remains unknown.The anti-apoptotic pathways utilized by human EC to resist LPS-induced apoptosis have yet to be elucidated. Human EC sensitivity to LPS-induced apoptosis is dependent on new protein or mRNA synthesis inhibition, suggesting that either an inducible or constitutively expressed protein is responsible for protection (30Hu X. Yee E. Harlan J.M. Wong F. Karsan A. Blood. 1998; 92: 2759-2765Crossref PubMed Google Scholar). LPS has previously been shown to up-regulate two cytoprotective proteins, the Bcl-2 homologue, A1, and the zinc-finger protein, A20, in EC (30Hu X. Yee E. Harlan J.M. Wong F. Karsan A. Blood. 1998; 92: 2759-2765Crossref PubMed Google Scholar). Overexpression of A1, A20, or Bcl-xL confers partial protection against LPS and cycloheximide (CHX)-induced apoptosis. There is doubt, however, concerning the physiological relevance of these overexpression studies. Similar to LPS, sensitization of human EC to TNF-α-induced apoptosis requires inhibition of gene expression. Further, overexpression of A1 or Bcl-xL protects EC against TNF-α and CHX induced-apoptosis (31Karsan A. Yee E. Harlan J.M. J. Biol. Chem. 1996; 271: 27201-27204Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar). The selective inhibition of A1 or Bcl-xL gene expression, however, fails to sensitize human EC to direct TNF-α-induced apoptosis, indicating that these proteins are not critical for protection (32Ackermann E.J. Taylor J.K. Narayana R. Bennett C.F. J. Biol. Chem. 1999; 274: 11245-11252Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). Another proposed mechanism of protection is through NF-κB-dependent gene expression of certain cytoprotective proteins, including cellular inhibitor of apoptosis proteins (cIAP) (33Wang C.Y. Mayo M.W. Korneluk R.G. Goeddel D.V. Baldwin Jr., A.S. Science. 1998; 281: 1680-1683Crossref PubMed Scopus (2562) Google Scholar). Selective blockade of the NF-κB signaling pathway by expression of a mutant IκB sensitizes EC to apoptosis induced by prolonged exposure to TNF-α, but not LPS (14Zen K. Karsan A. Stempien-Otero A. Yee E. Tupper J. Li X. Eunson T. Kay M.A. Wilson C.B. Winn R.K. Harlan J.M. J. Biol. Chem. 1999; 274: 28808-28815Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). This finding suggests that cell survival following LPS exposure is NF-κB-independent. In the present report, we have attempted to elucidate the mechanism of human EC resistance to LPS-induced apoptosis. Despite advances in anti-microbial therapy and overall medical care, Gram-negative bacterial sepsis remains a common, life-threatening event. The challenge of managing septic patients is compounded by the development of key vascular complications including, systemic vascular collapse, disseminated intravascular coagulation, and vascular leak syndromes (1Brigham K.L. Meyrick B. Am. Rev. Respir. Dis. 1986; 133: 913-927PubMed Google Scholar, 2Hinshaw L.B. Crit. Care Med. 1996; 24: 1072-1078Crossref PubMed Scopus (309) Google Scholar, 3Levi M. ten Cate H. van der Poll T. van Deventer S.J. JAMA. 1993; 270: 975-979Crossref PubMed Scopus (342) Google Scholar, 4Thijs L.G. de Boer J.P. de Groot M.C. Hack C.E. Intensive Care Med. 1993; 19: S8-15Crossref PubMed Scopus (87) Google Scholar, 5Ulevitch R.J. Cochrane C.G. Henson P.M. Morrison D.C. Doe W.F. J. Exp. Med. 1975; 142: 1570-1590Crossref PubMed Scopus (47) Google Scholar). A common denominator to all these complications is endothelial cell (EC)1 injury and/or dysfunction. Endotoxin or lipopolysaccharide (LPS), which resides in the outer membrane of Gram-negative bacteria, has been implicated as the causative agent responsible for EC dysfunction (1Brigham K.L. Meyrick B. Am. Rev. Respir. Dis. 1986; 133: 913-927PubMed Google Scholar, 5Ulevitch R.J. Cochrane C.G. Henson P.M. Morrison D.C. Doe W.F. J. Exp. Med. 1975; 142: 1570-1590Crossref PubMed Scopus (47) Google Scholar, 6Brandtzaeg P. Kierulf P. Gaustad P. Skulberg A. Bruun J.N. Halvorsen S. Sorensen E. J. Infect. Dis. 1989; 159: 195-204Crossref PubMed Scopus (467) Google Scholar, 7Ziegler E.J. McCutchan J.A. Fierer J. Glauser M.P. Sadoff J.C. Douglas H. Braude A.I. N. Engl. J. Med. 1982; 307: 1225-1230Crossref PubMed Scopus (735) Google Scholar). In the absence of non-endothelial cell-derived host mediator systems, LPS directly evokes numerous EC responses including: 1) up-regulation of adhesion molecules; 2) increased production of cytokines, nitric oxide, and tissue factor; 3) loss of monolayer integrity and barrier function; and 4) apoptosis (8Bannerman D.D. Goldblum S.E. Lab. Invest. 1999; 79: 1181-1199PubMed Google Scholar). In addition to a direct role, LPS stimulates the production of inflammatory cytokines, including interleukin (IL)-1β and tumor necrosis factor (TNF)-α, which also elicit an altered pathophysiological endothelial state (9Thijs L.G. Groeneveld A.B. Hack C.E. Curr. Top. Microbiol. Immunol. 1996; 216: 209-237PubMed Google Scholar). LPS-induced EC apoptosis has been observed both in vitro(10Bannerman D.D. Sathyamoorthy M. Goldblum S.E. J. Biol. Chem. 1998; 273: 35371-35380Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar, 11Choi K.B. Wong F. Harlan J.M. Chaudhary P.M. Hood L. Karsan A. J. Biol. Chem. 1998; 273: 20185-20188Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar, 12Frey E.A. Finlay B.B. Microb. Pathog. 1998; 24: 101-109Crossref PubMed Scopus (71) Google Scholar, 13Hoyt D.G. Mannix R.J. Rusnak J.M. Pitt B.R. Lazo J.S. Am. J. Physiol. 1995; 269: L171-L177PubMed Google Scholar, 14Zen K. Karsan A. Stempien-Otero A. Yee E. Tupper J. Li X. Eunson T. Kay M.A. Wilson C.B. Winn R.K. Harlan J.M. J. Biol. Chem. 1999; 274: 28808-28815Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar) and in vivo (15Fujita M. Kuwano K. Kunitake R. Hagimoto N. Miyazaki H. Kaneko Y. Kawasaki M. Maeyama T. Hara N. Int. Arch. Allergy Immunol. 1998; 117: 202-208Crossref PubMed Scopus (117) Google Scholar, 16Haimovitz-Friedman A. Cordon-Cardo C. Bayoumy S. Garzotto M. McLoughlin M. Gallily R. Edwards III, C.K. Schuchman E.H. Fuks Z. Kolesnick R. J. Exp. Med. 1997; 186: 1831-1841Crossref PubMed Scopus (379) Google Scholar). LPS directly induces apoptosis in bovine and ovine EC (10Bannerman D.D. Sathyamoorthy M. Goldblum S.E. J. Biol. Chem. 1998; 273: 35371-35380Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar, 12Frey E.A. Finlay B.B. Microb. Pathog. 1998; 24: 101-109Crossref PubMed Scopus (71) Google Scholar, 13Hoyt D.G. Mannix R.J. Rusnak J.M. Pitt B.R. Lazo J.S. Am. J. Physiol. 1995; 269: L171-L177PubMed Google Scholar, 17Hoyt D.G. Mannix R.J. Gerritsen M.E. Watkins S.C. Lazo J.S. Pitt B.R. Am. J. Physiol. 1996; 270: L689-L694PubMed Google Scholar). Sensitization of human EC to LPS-induced apoptosis requires the inhibition of either mRNA or protein synthesis (18Pohlman T.H. Harlan J.M. Cell Immunol. 1989; 119: 41-52Crossref PubMed Scopus (87) Google Scholar). This latter finding suggests that either a constitutively expressed protein with a relatively short half-life or an inducible protein is requisite for EC survival following LPS exposure. Similarly, two LPS-inducible cytokines, IL-1β and TNF-α, induce human EC apoptosis only when new gene expression is blocked (18Pohlman T.H. Harlan J.M. Cell Immunol. 1989; 119: 41-52Crossref PubMed Scopus (87) Google Scholar). Apoptosis has been implicated as an important mechanism of in vivo cell death following LPS exposure. Tissues and organs obtained from either patients who have died of sepsis and multi-organ failure (19Hotchkiss R.S. Swanson P.E. Freeman B.D. Tinsley K.W. Cobb J.P. Matuschak G.M. Buchman T.G. Karl I.E. Crit. Care Med. 1999; 27: 1230-1251Crossref PubMed Scopus (1042) Google Scholar) or animal models of endotoxemia and sepsis reveal enhanced apoptotic cell death (16Haimovitz-Friedman A. Cordon-Cardo C. Bayoumy S. Garzotto M. McLoughlin M. Gallily R. Edwards III, C.K. Schuchman E.H. Fuks Z. Kolesnick R. J. Exp. Med. 1997; 186: 1831-1841Crossref PubMed Scopus (379) Google Scholar, 20Hotchkiss R.S. Swanson P.E. Cobb J.P. Jacobson A. Buchman T.G. Karl I.E. Crit. Care Med. 1997; 25: 1298-1307Crossref PubMed Scopus (295) Google Scholar, 21Zhang X.M. Morikawa A. Takahashi K. Jiang G.Z. Kato Y. Sugiyama T. Kawai M. Fukada M. Yokochi T. Microbiol. Immunol. 1994; 38: 669-671Crossref PubMed Scopus (42) Google Scholar). The vascular endothelium is one tissue that is sensitive to LPS-induced apoptosis. In a murine model of sepsis, apoptotic EC have been detected in pulmonary capillaries (20Hotchkiss R.S. Swanson P.E. Cobb J.P. Jacobson A. Buchman T.G. Karl I.E. Crit. Care Med. 1997; 25: 1298-1307Crossref PubMed Scopus (295) Google Scholar). Intravenous administration of endotoxin into rabbits or rats induces EC death and detachment from the artery wall (22Gaynor E. Bouvier C. Spaet T.H. Science. 1970; 170: 986-988Crossref PubMed Scopus (89) Google Scholar, 23Reidy M.A. Schwartz S.M. Lab. Invest. 1983; 48: 25-34PubMed Google Scholar). In mice challenged with either LPS or TNF-α, disseminated EC apoptosis has been reported in the lung, thymus, and intestine (15Fujita M. Kuwano K. Kunitake R. Hagimoto N. Miyazaki H. Kaneko Y. Kawasaki M. Maeyama T. Hara N. Int. Arch. Allergy Immunol. 1998; 117: 202-208Crossref PubMed Scopus (117) Google Scholar, 16Haimovitz-Friedman A. Cordon-Cardo C. Bayoumy S. Garzotto M. McLoughlin M. Gallily R. Edwards III, C.K. Schuchman E.H. Fuks Z. Kolesnick R. J. Exp. Med. 1997; 186: 1831-1841Crossref PubMed Scopus (379) Google Scholar, 24Kawasaki M. Kuwano K. Hagimoto N. Matsuba T. Kunitake R. Tanaka T. Maeyama T. Hara N. Am. J. Pathol. 2000; 157: 597-603Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar). Finally, injection of a broad spectrum caspase inhibitor following LPS administration decreased EC apoptosis in the lung and improved survival in a murine model of acute lung injury (24Kawasaki M. Kuwano K. Hagimoto N. Matsuba T. Kunitake R. Tanaka T. Maeyama T. Hara N. Am. J. Pathol. 2000; 157: 597-603Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar). Together, these in vitro and in vivo studies indicate that the vascular endothelium is a key target of LPS-induced apoptosis. The mechanisms by which LPS activates apoptosis remain unknown. Efforts to elucidate LPS signaling pathways, apoptotic or otherwise, have been hampered by the lack of an identifiable membrane-bound receptor capable of signal transduction. Recently, Toll-like receptor (TLR)-4 has been identified in both cells of monocytic lineage and EC as the receptor responsible for LPS activation of the NF-κB signaling pathway (25Faure E. Equils O. Sieling P.A. Thomas L. Zhang F.X. Kirschning C.J. Polentarutti N. Muzio M. Arditi M. J. Biol. Chem. 2000; 275: 11058-11063Abstract Full Text Full Text PDF PubMed Scopus (506) Google Scholar, 26Hoshino K. Takeuchi O. Kawai T. Sanjo H. Ogawa T. Takeda Y. Takeda K. Akira S. J. Immunol. 1999; 162: 3749-3752PubMed Google Scholar, 27Lien E. Means T.K. Heine H. Yoshimura A. Kusumoto S. Fukase K. Fenton M.J. Oikawa M. Qureshi N. Monks B. Finberg R.W. Ingalls R.R. Golenbock D.T. J. Clin. Invest. 2000; 105: 497-504Crossref PubMed Scopus (683) Google Scholar). Interestingly, MyD88, a TLR-4-binding protein that has a requisite role in the downstream activation of NF-κB in EC, contains a death domain (DD) (28Kopp E.B. Medzhitov R. Curr. Opin. Immunol. 1999; 11: 13-18Crossref PubMed Scopus (586) Google Scholar, 29Zhang F.X. Kirschning C.J. Mancinelli R. Xu X.P. Jin Y. Faure E. Mantovani A. Rothe M. Muzio M. Arditi M. J. Biol. Chem. 1999; 274: 7611-7614Abstract Full Text Full Text PDF PubMed Scopus (535) Google Scholar). DD are conserved regions of amino acids, which facilitate protein-protein interaction. In the well characterized TNF pathway, the TNF receptor-binding protein, TRADD, recruits another adapter protein, FADD, through the interaction of their respective death domains. An additional conserved sequence in FADD, the death effector domain (DED), enables the recruitment of caspase 8, an upstream cysteine protease whose activation initiates a cascade of proteolytic events characteristic of apoptosis. We have previously reported that LPS-induced EC apoptosis is FADD-dependent (11Choi K.B. Wong F. Harlan J.M. Chaudhary P.M. Hood L. Karsan A. J. Biol. Chem. 1998; 273: 20185-20188Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar). Whether FADD is recruited to MyD88 following LPS stimulation via binding of their respective DD remains unknown. The anti-apoptotic pathways utilized by human EC to resist LPS-induced apoptosis have yet to be elucidated. Human EC sensitivity to LPS-induced apoptosis is dependent on new protein or mRNA synthesis inhibition, suggesting that either an inducible or constitutively expressed protein is responsible for protection (30Hu X. Yee E. Harlan J.M. Wong F. Karsan A. Blood. 1998; 92: 2759-2765Crossref PubMed Google Scholar). LPS has previously been shown to up-regulate two cytoprotective proteins, the Bcl-2 homologue, A1, and the zinc-finger protein, A20, in EC (30Hu X. Yee E. Harlan J.M. Wong F. Karsan A. Blood. 1998; 92: 2759-2765Crossref PubMed Google Scholar). Overexpression of A1, A20, or Bcl-xL confers partial protection against LPS and cycloheximide (CHX)-induced apoptosis. There is doubt, however, concerning the physiological relevance of these overexpression studies. Similar to LPS, sensitization of human EC to TNF-α-induced apoptosis requires inhibition of gene expression. Further, overexpression of A1 or Bcl-xL protects EC against TNF-α and CHX induced-apoptosis (31Karsan A. Yee E. Harlan J.M. J. Biol. Chem. 1996; 271: 27201-27204Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar). The selective inhibition of A1 or Bcl-xL gene expression, however, fails to sensitize human EC to direct TNF-α-induced apoptosis, indicating that these proteins are not critical for protection (32Ackermann E.J. Taylor J.K. Narayana R. Bennett C.F. J. Biol. Chem. 1999; 274: 11245-11252Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). Another proposed mechanism of protection is through NF-κB-dependent gene expression of certain cytoprotective proteins, including cellular inhibitor of apoptosis proteins (cIAP) (33Wang C.Y. Mayo M.W. Korneluk R.G. Goeddel D.V. Baldwin Jr., A.S. Science. 1998; 281: 1680-1683Crossref PubMed Scopus (2562) Google Scholar). Selective blockade of the NF-κB signaling pathway by expression of a mutant IκB sensitizes EC to apoptosis induced by prolonged exposure to TNF-α, but not LPS (14Zen K. Karsan A. Stempien-Otero A. Yee E. Tupper J. Li X. Eunson T. Kay M.A. Wilson C.B. Winn R.K. Harlan J.M. J. Biol. Chem. 1999; 274: 28808-28815Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). This finding suggests that cell survival following LPS exposure is NF-κB-independent. In the present report, we have attempted to elucidate the mechanism of human EC resistance to LPS-induced apoptosis.
Referência(s)