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Inhibition of Phosphatidylinositol 3-Kinase Sensitizes Vascular Endothelial Cells to Cytokine-initiated Cathepsin-dependent Apoptosis

2003; Elsevier BV; Volume: 278; Issue: 23 Linguagem: Inglês

10.1074/jbc.m212837200

ISSN

1083-351X

Autores

Lisa A. Madge, Jie‐Hui Li, Jaehyuk Choi, Jordan S. Pober,

Tópico(s)

Signaling Pathways in Disease

Resumo

In the presence of cycloheximide, tumor necrosis factor or interleukin-1 initiates caspase activation, loss of mitochondrial membrane potential (ΔΨ), DNA degradation, and nuclear condensation and fragmentation characteristic of apoptotic cell death in human vascular endothelial cells (EC). Inhibition of phosphatidylinositol 3-kinase (PI3K) by LY294002, but not inhibition of Akt by dominant-negative mutation, also sensitizes EC to cytokine-initiated apoptosis. Cytokine-initiated caspase activation is slower and comparatively less with LY294002 than with cycloheximide. Cycloheximide but not LY294002 decreases expression of c-FLIP (cellular FLICE inhibitory protein), an inhibitor of caspase-8 activation. The caspase inhibitor zVADfmk completely blocks caspase activation, DNA degradation, and nuclear fragmentation in both cases but only prevents loss of ΔΨ and cell death for cytokine plus cycloheximide treatment. In contrast, overexpression of Bcl-2 protects EC treated with cytokine plus LY294002 but not EC treated with cytokine plus cycloheximide. The cathepsin B inhibitor CA-074-Me prevents loss of ΔΨ, caspase activation, and cell death for EC treated with cytokine plus LY294002 but has no effect on EC treated with cytokine plus cycloheximide. Cathepsin B translocates from lysosomes to cytosol following treatment with LY294002 prior to the activation of caspases. These results suggest that inhibition of PI3K allows cytokines to activate a cathepsin-dependent, mitochondrial death pathway in which caspase activation is secondary, is not inhibited by c-FLIP, and is not essential for cell death. In the presence of cycloheximide, tumor necrosis factor or interleukin-1 initiates caspase activation, loss of mitochondrial membrane potential (ΔΨ), DNA degradation, and nuclear condensation and fragmentation characteristic of apoptotic cell death in human vascular endothelial cells (EC). Inhibition of phosphatidylinositol 3-kinase (PI3K) by LY294002, but not inhibition of Akt by dominant-negative mutation, also sensitizes EC to cytokine-initiated apoptosis. Cytokine-initiated caspase activation is slower and comparatively less with LY294002 than with cycloheximide. Cycloheximide but not LY294002 decreases expression of c-FLIP (cellular FLICE inhibitory protein), an inhibitor of caspase-8 activation. The caspase inhibitor zVADfmk completely blocks caspase activation, DNA degradation, and nuclear fragmentation in both cases but only prevents loss of ΔΨ and cell death for cytokine plus cycloheximide treatment. In contrast, overexpression of Bcl-2 protects EC treated with cytokine plus LY294002 but not EC treated with cytokine plus cycloheximide. The cathepsin B inhibitor CA-074-Me prevents loss of ΔΨ, caspase activation, and cell death for EC treated with cytokine plus LY294002 but has no effect on EC treated with cytokine plus cycloheximide. Cathepsin B translocates from lysosomes to cytosol following treatment with LY294002 prior to the activation of caspases. These results suggest that inhibition of PI3K allows cytokines to activate a cathepsin-dependent, mitochondrial death pathway in which caspase activation is secondary, is not inhibited by c-FLIP, and is not essential for cell death. Vascular endothelial cells (EC) 1The abbreviations used are: EC, endothelial cell; ΔΨ, mitochondrial membrane potential; CHX, cycloheximide; c-FLIP, FLICE inhibitory protein; DD, death domain; DISC, death-inducing signaling complex; FADD, Fas-associated death domain protein; IL-1, interleukin-1; NFκB, nuclear factor κB; PI3K, phosphatidylinositol 3-kinase; TNF, tumor necrosis factor; TNFR1, TNF receptor 1; TNFR2, TNF receptor 2; TRADD, TNF receptor-associated death domain protein; BH, Bcl-2 homology domain; FKHR, forkhead transcription factor; AMC, amidomethylcoumarin; DAPI, 4′,6-diamidino-2-phenylindole HCl; HA, hemagglutinin; PBS, phosphate-buffered saline; FACS, fluorescence-activated cell sorting; HUVEC, human umbilical vein endothelial cell; zVADfmk, N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone; CA-074-Me, [l-3-trans(propylcarbamoyl)oxirane-2-carboxyl]-l-isoleucyl-l-proline methylester. 1The abbreviations used are: EC, endothelial cell; ΔΨ, mitochondrial membrane potential; CHX, cycloheximide; c-FLIP, FLICE inhibitory protein; DD, death domain; DISC, death-inducing signaling complex; FADD, Fas-associated death domain protein; IL-1, interleukin-1; NFκB, nuclear factor κB; PI3K, phosphatidylinositol 3-kinase; TNF, tumor necrosis factor; TNFR1, TNF receptor 1; TNFR2, TNF receptor 2; TRADD, TNF receptor-associated death domain protein; BH, Bcl-2 homology domain; FKHR, forkhead transcription factor; AMC, amidomethylcoumarin; DAPI, 4′,6-diamidino-2-phenylindole HCl; HA, hemagglutinin; PBS, phosphate-buffered saline; FACS, fluorescence-activated cell sorting; HUVEC, human umbilical vein endothelial cell; zVADfmk, N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone; CA-074-Me, [l-3-trans(propylcarbamoyl)oxirane-2-carboxyl]-l-isoleucyl-l-proline methylester. are a principal target of the pro-inflammatory cytokines TNF and IL-1. Effects on the endothelium that contribute to the inflammatory response are largely dependent on gene transcription resulting in the expression of proteins controlling vasoregulation, leukocyte adhesion, leukocyte activation, and coagulation. In some cases TNF and IL-1 may also result in endothelial injury, a common feature in the pathogenesis of vascular leak, sepsis, and transplant rejection. The ability of TNF or IL-1 to cause endothelial injury may occur indirectly through the activation and recruitment of leukocytes or generation of thrombosis or may occur directly from the pro-apoptotic actions of these cytokines on EC. The direct pro-apoptotic action of TNF on various cell types generally results from the ligand-dependent assembly of a death-inducing signaling complex (DISC), so called for the ability of this complex to initiate caspase activation. For TNF, the formation of a DISC is dependent on ligand-binding to TNF receptor type I (TNFR-1, also designated CD120a), which leads to the recruitment of the cytosolic adapter protein TNFR-1 associated death domain protein (TRADD) (1Hsu H. Xiong J. Goeddel D.V. Cell. 1995; 81: 495-504Abstract Full Text PDF PubMed Scopus (1743) Google Scholar). The association between TNFR1 and TRADD involves the "death domains" (DD) of these proteins. DDs are homologous regions of ∼80 amino acids that mediate protein-protein interaction and are also found in other receptors such as Fas (CD95) (2Boldin M.P. Mett I.L. Varfolomeev E.E. Chumakov I. Shemer-Avni Y. Camonis J.H. Wallach D. J. Biol. Chem. 1995; 270: 387-391Abstract Full Text Full Text PDF PubMed Scopus (351) Google Scholar). TRADD may subsequently recruit Fas-associated death domain protein (FADD) through DD interactions (3Hsu H. Shu H.B. Pan M.G. Goeddel D.V. Cell. 1996; 84: 299-308Abstract Full Text Full Text PDF PubMed Scopus (1732) Google Scholar). FADD contains both a DD and a death effector domain, the latter of which can interact with either pro-caspase-8 (also known as FLICE) or with cellular FLICE inhibitory protein (c-FLIP) (4Boldin M.P. Goncharov T.M. Goltsev Y.V. Wallach D. Cell. 1996; 85: 803-815Abstract Full Text Full Text PDF PubMed Scopus (2109) Google Scholar, 5Irmler M. Thome M. Hahne M. Schneider P. Hofmann K. Steiner V. Bodmer J.L. Schroter M. Burns K. Mattmann C. Rimoldi D. French L.E. Tschopp J. Nature. 1997; 388: 190-195Crossref PubMed Scopus (2223) Google Scholar). FADD-associated pro-caspase-8 undergoes autocatalytic activation by proteolysis, liberating the active enzyme from the pro-form (6Muzio M. Stockwell B.R. Stennicke H.R. Salvesen G.S. Dixit V.M. J. Biol. Chem. 1998; 273: 2926-2930Abstract Full Text Full Text PDF PubMed Scopus (884) Google Scholar). c-FLIP may inhibit this process. Activated caspase-8 dissociates from the DISC and acts on various cytosolic substrates. For example, caspase-8 may proteolytically activate the effector caspase-3. Activated caspase-3, in turn, cleaves a variety of substrates, resulting in apoptotic cell death (7Stennicke H.R. Jurgensmeier J.M. Shin H. Deveraux Q. Wolf B.B. Yang X. Zhou Q. Ellerby H.M. Ellerby L.M. Bredesen D. Green D.R. Reed J.C. Froelich C.J. Salvesen G.S. J. Biol. Chem. 1998; 273: 27084-27090Abstract Full Text Full Text PDF PubMed Scopus (645) Google Scholar). Alternatively, caspase-8 may proteolytically activate a cytosolic protein called Bid (8Scaffidi C. Schmitz I. Zha J. Korsmeyer S.J. Krammer P.H. Peter M.E. J. Biol. Chem. 1999; 274: 22532-22538Abstract Full Text Full Text PDF PubMed Scopus (530) Google Scholar). Bid is a pro-apoptotic Bcl-2 family member containing a single Bcl-2 homology (BH) domain designated BH3. Proteolytically activated forms of "BH3-only" family members, such as Bid and Bad, bind to mitochondrial-associated "BH1–3" or "multidomain" proteins, such as Bax and Bak, causing supramolecular openings of the outer mitochondrial membrane (9Ottilie S. Diaz J.L. Horne W. Chang J. Wang Y. Wilson G. Chang S. Weeks S. Fritz L.C. Oltersdorf T. J. Biol. Chem. 1997; 272: 30866-30872Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar, 10Kuwana T. Smith J.J. Muzio M. Dixit V. Newmeyer D.D. Kornbluth S. J. Biol. Chem. 1998; 273: 16589-16594Abstract Full Text Full Text PDF PubMed Scopus (335) Google Scholar, 11Gross A. Jockel J. Wei M.C. Korsmeyer S.J. EMBO J. 1998; 17: 3878-3885Crossref PubMed Scopus (966) Google Scholar, 12Kuwana T. Mackey M.R. Perkins G. Ellisman M.H. Latterich M. Schneiter R. Green D.R. Newmeyer D.D. Cell. 2002; 111: 331-342Abstract Full Text Full Text PDF PubMed Scopus (1225) Google Scholar). These openings allow the release of cytochrome c from the mitochondria (13Luo X. Budihardjo I. Zou H. Slaughter C. Wang X. Cell. 1998; 94: 481-490Abstract Full Text Full Text PDF PubMed Scopus (3076) Google Scholar). Within the cytosol, cytochrome c associates with Apaf-1, and this complex further recruits pro-caspase-9 forming an assembly sometimes called an apoptosome. The apoptosome mediates ATP-dependent autocatalytic processing of caspase-9 and activated caspase-9, like caspase-8, can catalyze the proteolytic activation of caspase-3 resulting in apoptotic cell death (14Hu Y. Benedict M.A. Ding L. Nunez G. EMBO J. 1999; 18: 3586-3595Crossref PubMed Scopus (409) Google Scholar). Thus caspase-8-mediated cleavage of Bid activates an amplification pathway for mitochondrial-dependent activation of caspase-3. Mitochondrial openings may also release other proteins, such as apoptosis-inducing factor that can initiate caspase-independent cell death (15Joza N. Susin S.A. Daugas E. Stanford W.L. Cho S.K. Li C.Y. Sasaki T. Elia A.J. Cheng H.Y. Ravagnan L. Ferri K.F. Zamzami N. Wakeham A. Hakem R. Yoshida H. Kong Y.Y. Mak T.W. Zuniga-Pflucker J.C. Kroemer G. Penninger J.M. Nature. 2001; 410: 549-554Crossref PubMed Scopus (1152) Google Scholar). The requirement for the Bid/cytochrome c/Apaf1 amplification pathway differs among various cell types and correlates with the extent to which active caspase-8 is generated by the DISC (8Scaffidi C. Schmitz I. Zha J. Korsmeyer S.J. Krammer P.H. Peter M.E. J. Biol. Chem. 1999; 274: 22532-22538Abstract Full Text Full Text PDF PubMed Scopus (530) Google Scholar, 16Scaffidi C. Fulda S. Srinivasan A. Friesen C. Li F. Tomaselli K.J. Debatin K.M. Krammer P.H. Peter M.E. EMBO J. 1998; 17: 1675-1687Crossref PubMed Scopus (2624) Google Scholar). DISC activity may be positively regulated by the expression levels of FADD and pro-caspase-8 (17Li J.H. Kluger M.S. Madge L.A. Zheng L. Bothwell A.L. Pober J.S. Am. J. Pathol. 2002; 161: 1485-1495Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar) or negatively regulated by the levels of c-FLIP (5Irmler M. Thome M. Hahne M. Schneider P. Hofmann K. Steiner V. Bodmer J.L. Schroter M. Burns K. Mattmann C. Rimoldi D. French L.E. Tschopp J. Nature. 1997; 388: 190-195Crossref PubMed Scopus (2223) Google Scholar). IL-1, like TNF, initiates the activation of signal transduction cascades by the recruitment of adapter proteins to its receptor. In EC, IL-1 may also initiate apoptotic cell death. To date, the precise components of the IL-1 receptor-associated DISC have not been defined. A number of adapter proteins involved in IL-1 signal transduction, namely MyD88 and IRAK (18Wesche H. Henzel W.J. Shillinglaw W. Li S. Cao Z. Immunity. 1997; 7: 837-847Abstract Full Text Full Text PDF PubMed Scopus (918) Google Scholar), contain DDs and are possible mediators of caspase recruitment and activation. Most untransformed cell types are not sensitive to the pro-apoptotic actions of TNF or IL-1, unless mRNA translation or protein synthesis is blocked. This observation has been explained by the capacity of TNF to stimulate the activation of NFκB, resulting in the up-regulation of anti-apoptotic gene products such as c-FLIP, XIAP, c-IAP 1, and c-IAP 2 (19Kreuz S. Siegmund D. Scheurich P. Wajant H. Mol. Cell. Biol. 2001; 21: 3964-3973Crossref PubMed Scopus (503) Google Scholar, 20Micheau O. Lens S. Gaide O. Alevizopoulos K. Tschopp J. Mol. Cell. Biol. 2001; 21: 5299-5305Crossref PubMed Scopus (685) Google Scholar, 21Martin S.J. Cell. 2002; 109: 793-796Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). The expression levels of several anti-apoptotic genes, such as c-FLIP and IAP 1 are also regulated by the proteasome (22Bannerman D.D. Tupper J.C. Ricketts W.A. Bennett C.F. Winn R.K. Harlan J.M. J. Biol. Chem. 2001; 276: 14924-14932Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar, 23Yang Y. Fang S. Jensen J.P. Weissman A.M. Ashwell J.D. Science. 2000; 288: 874-877Crossref PubMed Scopus (866) Google Scholar). In the presence of cycloheximide (CHX), levels of c-FLIP are rapidly diminished, favoring DISC-dependent activation of caspase-8 (19Kreuz S. Siegmund D. Scheurich P. Wajant H. Mol. Cell. Biol. 2001; 21: 3964-3973Crossref PubMed Scopus (503) Google Scholar, 22Bannerman D.D. Tupper J.C. Ricketts W.A. Bennett C.F. Winn R.K. Harlan J.M. J. Biol. Chem. 2001; 276: 14924-14932Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). Reduction of c-FLIP by antisense oligonucleotides mimics the effect of CHX and similarly sensitizes cells to death (22Bannerman D.D. Tupper J.C. Ricketts W.A. Bennett C.F. Winn R.K. Harlan J.M. J. Biol. Chem. 2001; 276: 14924-14932Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). Although apoptosis is generally associated with caspase activation, either through a DISC or through an apoptosome, caspase-independent cell death has been observed with the generation of apoptotic-like features in a variety of cell types (24Deas O. Dumont C. MacFarlane M. Rouleau M. Hebib C. Harper F. Hirsch F. Charpentier B. Cohen G.M. Senik A. J. Immunol. 1998; 161: 3375-3383PubMed Google Scholar, 25Foghsgaard L. Wissing D. Mauch D. Lademann U. Bastholm L. Boes M. Elling F. Leist M. Jaattela M. J. Cell Biol. 2001; 153: 999-1010Crossref PubMed Scopus (562) Google Scholar, 26Luschen S. Ussat S. Scherer G. Kabelitz D. Adam-Klages S. J. Biol. Chem. 2000; 275: 24670-24678Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). In many instances, these variant forms of apoptosis are mediated by non-caspase proteases such as the cysteine protease cathepsin family, calpains, serine proteases, or the proteasome complex. Of particular interest, the activation of cathepsin B has been shown to play a central role in the generation on TNF-mediated cell death in fibrosarcoma cells (25Foghsgaard L. Wissing D. Mauch D. Lademann U. Bastholm L. Boes M. Elling F. Leist M. Jaattela M. J. Cell Biol. 2001; 153: 999-1010Crossref PubMed Scopus (562) Google Scholar). Furthermore, TNF-mediated apoptosis has been shown to be strongly reduced in hepatocytes from cathepsin B-deficient mice (27Guicciardi M.E. Miyoshi H. Bronk S.F. Gores G.J. Am. J. Pathol. 2001; 159: 2045-2054Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar). This pathway has not been described in a normal (untransformed) human cell type. While most TNF activities on EC result in inflammation and/or apoptosis, we have shown in EC that TNF and IL-1 also activate the anti-apoptotic phosphatidylinositol 3-kinase/Akt pathway (28Madge L.A. Pober J.S. J. Biol. Chem. 2000; 275: 15458-15465Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar). PI3K converts plasma membrane phosphatidylinositol 4,5-bisphosphate into phosphatidylinositol 3,4,5-trisphosphate, catalyzing the recruitment of several enzymes, such as PI3K-dependent protein kinase and Akt to the plasma membrane. Akt is a serine/threonine kinase that exerts an anti-apoptotic action by the phosphorylation of a number of substrates containing the phosphorylation consensus RXRXX(S/T). Akt-mediated phosphorylation inactivates the pro-apoptotic Bcl-2 homologue Bad (29Datta S.R. Dudek H. Tao X. Masters S. Fu H. Gotoh Y. Greenberg M.E. Cell. 1997; 91: 231-241Abstract Full Text Full Text PDF PubMed Scopus (4936) Google Scholar), the apoptosis-initiating enzyme caspase-9 (30Cardone M.H. Roy N. Stennicke H.R. Salvesen G.S. Franke T.F. Stanbridge E. Frisch S. Reed J.C. Science. 1998; 282: 1318-1321Crossref PubMed Scopus (2728) Google Scholar), and the forkhead family transcription factor FKHRL1 (31Brunet A. Bonni A. Zigmond M.J. Lin M.Z. Juo P. Hu L.S. Anderson M.J. Arden K.C. Blenis J. Greenberg M.E. Cell. 1999; 96: 857-868Abstract Full Text Full Text PDF PubMed Scopus (5412) Google Scholar), which mediates transcription of pro-apoptotic gene products. In some cells Akt may regulate the activity of NFκB either through direct phosphorylation and activation of IKKα (32Ozes O.N. Mayo L.D. Gustin J.A. Pfeffer S.R. Pfeffer L.M. Donner D.B. Nature. 1999; 401: 82-85Crossref PubMed Scopus (1896) Google Scholar) or through regulation of the transactivation capacity of Rel A (33Sizemore N. Leung S. Stark G.R. Mol. Cell. Biol. 1999; 19: 4798-4805Crossref PubMed Google Scholar). We have shown that activation of PI3K in EC does not contribute to the activation of NFκB or have any significant effect on NFκB-dependent inflammatory responses (28Madge L.A. Pober J.S. J. Biol. Chem. 2000; 275: 15458-15465Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar). Similarly, Akt is reported not to have any effect of NFκB activation in HeLa cells (34Delhase M. Li N. Karin M. Nature. 2000; 406: 367-368Crossref PubMed Scopus (121) Google Scholar). In the present study we have examined the role of PI3K and Akt activation in the regulation of apoptosis induced by TNF and IL-1. To do so, we either inhibited PI3K with LY294002 or inhibited Akt by retroviral transduction with an inactive (K179M), dominant-negative form of Akt. We report that inhibition of PI3K but not of Akt sensitizes EC to the apoptotic actions of TNF and IL-1 and that the cell death caused by this pathway could not be blocked by caspase inhibition with zVADfmk but instead appears to be mediated through cathepsin B. Cell Culture—Human umbilical vein EC were isolated from discarded tissue in accordance with an approved protocol by Yale University Human Investigations Committee and serially cultured on gelatin (J. T. Baker Inc., Phillipsburg, NJ)-coated tissue culture plastic (Falcon, Lincoln Park, NJ) in Medium 199 (M199) supplemented with 20% fetal calf serum, 200 μm l-glutamine (all from Invitrogen, Grand Island, NY), 50 μg/ml EC growth factor (ECGF) (Collaborative Biomedical Products, Bedford, MA), 100 μg/ml porcine heparin (Sigma Chemical Co., St. Louis, MO), 100 units/ml penicillin, and 100 μg/ml streptomycin (Invitrogen). All experiments were performed using EC at passage 2 or 3. Such sub-cultured EC uniformly display CD31 and von Willebrand factor and are free of CD45+-contaminating leukocytes. For replating assays floating EC were harvested, washed with Hanks' balance salt solution, and re-seeded onto gelatin-coated plates. EC that remained substrate-attached were similarly re-plated following harvesting with trypsin (Invitrogen). Materials—Recombinant human TNF was purchased from R&D, and IL-1 was purchased from Peprotech Inc (Rocky Hill, NJ). LY294002, CHX, and Z-Arg-Arg-amidomethylcoumarin (Z-Arg-Arg-AMC) were purchased from Calbiochem (San Diego, CA). Propidium iodide, Hoescht reagent, 4′,6-diamidino-2-phenylindole HCl (DAPI), JC-1, calcein-AM, and LysoTracker red were purchased from Molecular Probes (Eugene, OR). RNase A and digitonin were purchased from Sigma. The cathepsin B inhibitor CA-074-Me was purchased from Peptides International (Louisville, KY). Complete protease inhibitor mixture tablets and Pefabloc were purchased from Roche Applied Science (Indianapolis, IN). Mouse anti-FLIP antibody was a gift from Dr. Peter Krammer (DFKZ, Heidelberg, Germany). Rabbit anti-Bid antibody was purchased from BD Pharmingen (San Jose, CA). Mouse anti-cathepsin B was purchased from Oncogene Research Products (San Diego, CA). CaspaTag fluorescein broad range (VAD), caspase-3 (DEVD), caspase-8 (LETD), and caspase-9 (LEHD) activity assay kits were purchased from Serologicals (Norcross, GA). Mouse anti-Bax antibody was purchased from Transduction Laboratories (San Jose, CA). Rabbit anti-FKHR and phospho-FKHR antibodies were purchased from Cell Signaling (Beverly, MA). Mouse anti-hemagglutinin (HA) was purchased from Roche Applied Science (Indianapolis, IN). Horseradish peroxidase-conjugated secondary antibodies for Western blotting were purchased from Jackson ImmunoResearch (Westgrove, PA). Immunoblotting—For immunoblots, each well of a six-well plate containing a confluent HUVEC monolayer was washed twice in ice-cold PBS and lysed by the addition of 100 μl of lysis buffer (50 mm Tris-Cl, pH 6.8, 150 mm NaCl, and 1% Triton X-100) supplemented with Pefabloc (1 mm) and complete protease inhibitor mixture. For the measurement of phospho-proteins, NaF (10 mm) and Na3VO4 (1 mm) were also included in the lysis buffer to reduce phosphatase activity. After 20 min on ice, lysates were harvested by scraping. Where indicated, detached EC were harvested by centrifugation, washed in PBS, and pooled with the lysate of the attached EC from the same sample. For each sample, an equal amount of protein was separated by SDS-PAGE (35Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207012) Google Scholar) then transferred electrophoretically to a polyvinylidene difluoride membrane (Immobilon P, Millipore, Milford, MA) and immunoblotted with primary and horseradish peroxidase-conjugated secondary antibodies. Detection of the bound antibody by enhanced chemiluminescence was performed according to the manufacturer's instructions (Pierce Chemical Co., Rockford, IL). Cell Cycle and Hypodiploid DNA Analysis—EC grown to confluence on 12-well plates were treated as described in the text. At the indicated time after treatment, floating EC were collected and pooled with residual attached EC suspended by trypsin treatment. The pooled EC were washed once in PBS and fixed by resuspension in 70% ethanol for 15 min. After fixation, EC were washed once more in PBS before incubation in PBS containing propidium iodide (50 μg/ml) and RNase A (1 mg/ml) for 0.25–2 h. The DNA content of EC was then determined by FACS analysis using Cell Quest software (FACSort, BD Biosciences, San Jose, CA). DAPI Staining of EC—EC were grown to confluence on gelatin-coated 12-well plates and treated as described in the text. After treatment, attached EC were harvested with trypsin and combined with floating EC harvested from the same sample. Cells were washed in PBS, and ∼1 × 105 cells were adhered to a glass coverslip by spinning in a cyto-centrifuge (Shandon, Pittsburgh, PA). Slides were air-dried and dipped in a chamber containing MeOH and DAPI (1 μg/ml). After rinsing in PBS, a drop of Gel Mount (Biomeda Corp., Foster City, CA) and a coverslip was placed over the cells. Specimens were examined by immunofluorescence microscopy using a Nikon diaphot microscope with a 360-nm filter. Quantitation of EC Adherence and Replating—To quantify the number of cells that remained adherent, EC plated on gelatin-coated 96-well plates were treated as indicated in the text. After experimental manipulation the medium was removed and cells were washed twice in PBS. The residual attached cells were fixed and stained by the addition of 70% ethanol containing 100 μg/ml Hoescht 33258 reagent (Molecular Probes, Eugene, OR) for 30 min at room temperature. Cells were again washed twice with PBS, and the residual fluorescence was recorded (λex = 360 nm, λem = 460 nm) using a fluorescence plate reader (Perspective Biosystems Inc, Framingham, MA). To assess the viability of detached versus adherent cells, EC were grown to confluency in 12-well plates and treated as indicated in the text. Detached EC were harvested, washed in Hanks' balanced salt solution, and re-seeded onto gelatin-coated plates. EC that remained substrate-attached were similarly re-plated following harvest with trypsin. Viability was assessed as replating efficiency 18 h later quantified by Hoescht staining as above. Caspase Activity Assays—For experimental manipulation EC were plated on 12-well plates and treated at confluency as indicated in the text. After described treatment the CaspaTag peptide (FAM-VAD-fmk for broad range caspase activity, FAM-LETD-fmk for caspase-8, FAM-LEHD-fmk for caspase-9, or FAM-DEVD-fmk for caspase-3) was added to each well and incubated a further 1 h according to the manufacturer's instructions. Subsequent to incubation with the peptide, floating EC were harvested and combined with attached EC from the same well harvested with trypsin. Caspase activity was demonstrated by the generation of a second peak or shoulder on FL-1 that results from peptide binding to active caspase by FACS. Mitochondrial Membrane Potential (ΔΨ) Analysis—After experimental manipulation of EC seeded on 12-well plates, floating EC were harvested by centrifugation and combined with remaining substrate-attached EC harvested with trypsin. The pooled EC were washed 1× in PBS containing 1% bovine serum albumin before resuspension in 200 μl of PBS/bovine serum albumin containing JC-1 (10 μg/ml). After 15 min of incubation at 37 °C, EC were washed, re-suspended in PBS, and analyzed by FACS. Retroviral Transduction—HA-tagged murine K179M Akt (a gift from Dr. W. Sessa, Yale University) was Topo®-cloned using EcoRI and NotI into the retroviral LZRS expression vector (a gift from Dr. A. L. M. Bothwell, Yale University), and the construct was verified by sequencing. The caspase-resistant Bcl-2 retroviral construct (a gift from A. L. M. Bothwell) has been described previously (36Zheng L. Dengler T.J. Kluger M.S. Madge L.A. Schechner J.S. Maher S.E. Pober J.S. Bothwell A.L. J. Immunol. 2000; 164: 4665-4671Crossref PubMed Scopus (66) Google Scholar). The amphotropic Phoenix packaging cell line was transfected with either the empty vector LZRS, LZRS-K179M Akt, or LZRS-Bcl2 using LipofectAMINE (Invitrogen) and selected for gene expression 24 h after transfection using puromycin (1 μg/ml). Puromycin-resistant cells were used to derive conditioned medium to provide a retroviral stock for HUVEC transduction. For transduction of primary HUVECs, M199 containing ECGF was removed, and cells were washed and incubated 5–8 h with retroviral conditioned media containing Polybrene (8 μg/ml, Sigma). After incubation, retrovirus was removed and replaced with normal growth medium overnight. The transduction process was repeated a further three times with intermittent cell passage as required. Using this protocol the percentage of HUVECs expressing the transgene is routinely >95%. Preparation of Cytosolic Extracts for the Analysis of Cathepsin B—Measurement of cytosolic cathepsin B was determined using methodology similar to Foghsgaard et al. (25Foghsgaard L. Wissing D. Mauch D. Lademann U. Bastholm L. Boes M. Elling F. Leist M. Jaattela M. J. Cell Biol. 2001; 153: 999-1010Crossref PubMed Scopus (562) Google Scholar). Endothelial cells were pretreated with LY294002 (50 μm) for 3 h in complete M199 prior to the addition of cytokine for a further 3 h. After treatment media were removed and cells were washed twice in PBS prior to the addition of extraction buffer (50 μg/ml digitonin, 250 mm sucrose, 20 mm Hepes, 10 mm KCl, 1.5 mm MgCl2, 1 mm EDTA, 1 mm EGTA, 1 mm Pefabloc, pH 7.5) and incubation on ice for 20 min. (The conditions that allow for the selective permeabilization of the plasma membrane by digitonin without perturbation of lysosomes were determined in preliminary experiments using EC pre-loaded both with calcein-AM and LysoTracker red.) After incubation, the cytosolic extract was collected. Samples were analyzed for cathepsin B either by Western blotting or with a cathepsin B activity assay. Measurement of Cathepsin B Activity—A 50-μl volume of cytosolic extract was added to an equal volume of cathepsin reaction buffer (50 mm sodium acetate, 4 mm EDTA, 8 mm dithiothreitol, 1 mm Pefabloc, pH 6.0). Cathepsin B activity was measured by the addition of 20 μmZ-Arg-Arg-AMC (Calbiochem). Liberated AMC was measured (λex = 360 nm, λem = 460 nm) using a fluorescence plate reader immediately following the addition of the peptide substrate (T0) and following a 60 min incubation at 37 °C (T60). Activity was determined by subtracting the background activity at T0 from activity at T60 and correcting for the amount of protein in each sample. TNF and IL-1 Mediate Nuclear Condensation and EC Death under Conditions Where Either Protein Synthesis Is Inhibited or PI3K Activation Is Blocked—Many cell types are not sensitive to the pro-apoptotic actions of TNF or IL-1 unless RNA or protein synthesis is blocked. As previously described (37Slowik M.R. De Luca L.G. Min W. Pober J.S. Circ. Res. 1996; 79: 736-747Crossref PubMed Scopus (44) Google Scholar), treatment

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