A constitutive cytoprotective pathway protects endothelial cells from lipopolysaccharide-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-alpha or interleukin-1beta 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 Gramnegative 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-␣ or interleukin-1␤ 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.
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 (1)(2)(3)(4)(5). A common denominator to all these complications is endothelial cell (EC) 1 injury and/or dysfunction. Endotoxin or lipo-polysaccharide (LPS), which resides in the outer membrane of Gram-negative bacteria, has been implicated as the causative agent responsible for EC dysfunction (1,(5)(6)(7). 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 (8). In addition to a direct role, LPS stimulates the production of inflammatory cytokines, including interleukin (IL)-1␤ and tumor necrosis factor (TNF)-␣, which also elicit an altered pathophysiological endothelial state (9).
LPS-induced EC apoptosis has been observed both in vitro (10 -14) and in vivo (15,16). LPS directly induces apoptosis in bovine and ovine EC (10,12,13,17). Sensitization of human EC to LPS-induced apoptosis requires the inhibition of either mRNA or protein synthesis (18). 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-1␤ and TNF-␣, induce human EC apoptosis only when new gene expression is blocked (18).
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 (19) or animal models of endotoxemia and sepsis reveal enhanced apoptotic cell death (16,20,21). The vascular endothelium is one tissue that is sensitive to LPSinduced apoptosis. In a murine model of sepsis, apoptotic EC have been detected in pulmonary capillaries (20). Intravenous administration of endotoxin into rabbits or rats induces EC death and detachment from the artery wall (22,23). In mice challenged with either LPS or TNF-␣, disseminated EC apoptosis has been reported in the lung, thymus, and intestine (15,16,24). 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 (24). 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 trans-duction. 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-B signaling pathway (25)(26)(27). Interestingly, MyD88, a TLR-4-binding protein that has a requisite role in the downstream activation of NF-B in EC, contains a death domain (DD) (28,29). 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 (11). 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 (30). LPS has previously been shown to up-regulate two cytoprotective proteins, the Bcl-2 homologue, A1, and the zinc-finger protein, A20, in EC (30). Overexpression of A1, A20, or Bcl-x L 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-␣induced apoptosis requires inhibition of gene expression. Further, overexpression of A1 or Bcl-x L protects EC against TNF-␣ and CHX induced-apoptosis (31). The selective inhibition of A1 or Bcl-x L gene expression, however, fails to sensitize human EC to direct TNF-␣-induced apoptosis, indicating that these proteins are not critical for protection (32). Another proposed mechanism of protection is through NF-B-dependent gene expression of certain cytoprotective proteins, including cellular inhibitor of apoptosis proteins (cIAP) (33). Selective blockade of the NF-B signaling pathway by expression of a mutant IB sensitizes EC to apoptosis induced by prolonged exposure to TNF-␣, but not LPS (14). This finding suggests that cell survival following LPS exposure is NF-B-independent. In the present report, we have attempted to elucidate the mechanism of human EC resistance to LPS-induced apoptosis.
Detection of Apoptosis-EC viability was measured with Alamar Blue vital dye (35,36), and apoptosis was assayed by the detection of poly(ADP-ribose) polymerase (PARP) cleavage (37,38) and histone release into the cytoplasm (39). To assess viability, EC were seeded into 96-well plates at a density of 20,000 cells/well. After 48 h in culture, cells were subjected to various experimental conditions and 3 h prior to the end of treatment, Alamar Blue (10% final concentration; BIO-SOURCE International, Inc., Camarillo, CA) was added. Monolayers were assayed in a Cytofluor Series 4000 fluorescence plate reader (Perseptive Biosystems Inc., Framingham, MA) at 530 nm excitation and 590 nm emission and viability expressed in arbitrary fluorescent units.
For the detection of PARP cleavage, EC were scraped into ice-cold PBS, briefly centrifuged, and an equal number of EC resuspended in lysis buffer (62.5 mM Tris-HCl (pH 6.8), 6 M urea, 10% glycerol, 2% sodium dodecyl sulfate, 5% ␤-mercaptoethanol, and .002% bromphenol blue). EC lysates were then sonicated on ice for 15 s, incubated for 15 min at 65°C, loaded on a 4 -20% Tris-glycine gradient gel, and transferred to PVDF membrane. After blocking, the blots were incubated with anti-PARP antibody (1 g/ml; Biomol Research Laboratories, Inc., Plymouth Meeting, MA), subsequently incubated with HRP-conjugated anti-mouse IgG, and developed as above. For the quantitation of apoptosis, EC were seeded into 96-well plates at a density of 20,000 cells/ well, cultured for 48 h, treated, and histone release measured with the Cell Death Detection ELISA Plus assay according to the manufacturer's instructions (Roche Molecular Biochemicals). The plates were analyzed at 405 and 450 nm (reference wavelength) on a microplate reader (Bio-Tec Instruments, Inc., Winooski, VT). Background readings defined as EC exposed to medium alone were subtracted from each experimental treatment.
Vascular Cell Adhesion Molecule-1 (VCAM-1) Enzyme-linked Immunosorbent Assay-EC were seeded into 96-well plates at a density of 20,000 cells/well and cultured for 48 h. Following treatment, cells were washed twice with RPMI media supplemented with 2.5% bovine calf serum and incubated with 5 g/ml anti-VCAM-1 monoclonal antibody (clone 4B9) for 1 h at 37°C. Monolayers were washed as above, fixed for 10 min with 0.05% glutaraldehyde in PBS, washed again, and incubated with HRP-conjugated anti-mouse IgG (1 g/ml) (Transduction Laboratories) for 1 h at 37°C. EC were then washed five times with PBS, developing solution (1 mg/ml o-phenylaminediamine in 0.1 M sodium citrate (pH 4.5) with 0.003% hydrogen peroxide) added for 10 min at room temperature, and plates analyzed at 490 nm on a microplate reader (Bio-Tec Instruments, Inc.).
Adenoviral Transduction-The IB␣ mutant (IB␣M) and ␤-galactosidase recombinant adenoviruses were gifts of Dr. C. B. Wilson (University of Washington, Seattle, WA). The IB␣M (S32A/S36A) cDNA was generously provided by Drs. J. DiDonato and M. Karin (University of California, San Diego, CA) (40). Construction and purification of IB␣M and control (␤-galactosidase) recombinant adenovirus were performed as previously described (14). For adenoviral transduction, EC were seeded into 96-well plates at a density of 8,000 cells/well for 24 h, washed once with medium, and incubated for 72 h at a multiplicity of infection of 1000 with control or IB␣M adenovirus in complete EC medium. EC were washed once with complete medium and subjected to experimental treatment.
Oligonucleotide Design and Synthesis-2Ј-O-Methoxyethyl/2Јdeoxynucleotide chimeric antisense oligonucleotides (32,41) were used in all experiments. Chimeric oligonucleotides were used to support an RNase H-dependent mechanism of action, which results in a selective loss of target mRNA (42). All oligonucleotides were synthesized and purified as described previously (32,41). The c-FLIP and Mcl-1 antisense oligonucleotides, respectively, were identified by screening a series of different antisense oligonucleotides designed to hybridize to their respective targets using quantitative reverse transcription-polymerase chain reaction and Northern blot assays (43). ISIS 23296 and ISIS 20408 were found to be the most effective inhibitors of c-FLIP and Mcl-1 expression, respectively. The control antisense oligonucleotide contained eight or four mismatches, as compared with FLIP and Mcl-1 antisense oligonucleotides, respectively. Sequence of the oligonucleotides used are shown in Table I.
Oligonucleotide Transfection of EC-EC were seeded into 60-mm dishes and grown to ϳ80% confluence. EC were rinsed twice with Opti-MEM I (Life Technologies, Inc.) followed by incubation with 200 nM oligonucleotides premixed with 10 g/ml Lipofectin reagent (Life Technologies, Inc.) in Opti-MEM I. After a 4-h incubation at 37°C, the oligonucleotide solution was replaced with normal growth medium and EC allowed to recover for 8 h. EC were then treated with LPS in the presence or absence of CHX for 12 h and assayed for protein expression and apoptosis.
Statistical Methods-Analyses of variance was used to compare the mean responses among experimental and control groups. The Tukey post hoc comparison test was used to determine between which groups significant differences existed. All statistical analyses were performed using GraphPad Prism version 3.00 for Windows 95 (GraphPad Software, Inc., San Diego, CA). A p value of Ͻ0.05 was considered significant.

Time-dependent Effect of LPS-induced EC Apoptosis in the
Absence of New Protein Synthesis-Exposure to LPSϩCHX induced EC death in a time-dependent manner (Fig. 1A). An 8-h stimulus-to-response lag time was observed following LPSϩCHX treatment. EC exposed to CHX alone demonstrated no change in viability over this time period, consistent with the ability of CHX to inhibit de novo protein synthesis and cell proliferation without inducing EC death. Increased metabolic activity in EC exposed to medium or LPS alone reflected continued EC proliferation over time. To confirm that the decreased EC viability following administration of LPSϩCHX was due to apoptosis, cleavage of PARP and histone release into the cytoplasm were assayed (Fig. 1, B and C). PARP is a known substrate of caspases, a family of highly specific proteases activated during apoptosis (38). Western blot analysis revealed PARP cleavage only in lysates derived from EC exposed to LPS in combination with CHX. Consistent with these data, Hu et al. (30) reported that EC treated with LPS alone or CHX alone demonstrate no evidence of apoptosis, but upon co-administration of both LPS and CHX, cleavage of DNA into repeating oligonucleosomal fragments of 180 base pairs, a hallmark of apoptosis, was observed. Cleavage of PARP was timedependent and observed at exposures of Ն12 h. Another hallmark of apoptosis is the release of histones into the cytoplasm (39). Using a highly sensitive ELISA-based assay, the presence of cytosolic mono-and oligonucleosomes were only detected in EC exposed to both LPS and cycloheximide for Ն12 h (Fig. 1C), consistent with the detection of PARP cleavage.
The requisite inhibition of de novo protein synthesis necessary for EC sensitization to LPS-induced apoptosis has been reported for other inflammatory stimuli as well, including TNF-␣ and IL-1␤ (14). Further, LPS or TNF-␣ in the presence of actinomycin D, an inhibitor of mRNA synthesis, induces EC apoptosis suggesting that inhibition of gene expression regardless of whether it occurs at the protein or mRNA level is sufficient for sensitization (18). The ability of LPS to induce EC apoptosis only in the absence of new gene expression implies that either an inducible or constitutively expressed cytoprotective protein is required for EC resistance to LPS-evoked cell death.
Western Blot Analysis of Anti-apoptotic Proteins following EC Exposure to LPS and CHX-That inhibition of de novo protein synthesis sensitizes EC to LPS-induced apoptosis suggests that either a rapidly degraded constitutive protein and/or an inducible protein confer resistance under physiological conditions. We, therefore, screened EC exposed to medium, LPS, CHX, or LPSϩCHX for changes in the expression of known anti-apoptotic proteins constitutively expressed in human EC, including Akt (44)

Endothelial Cytoprotection against LPS-induced Apoptosis
shown to decrease in the presence of CHX ( Fig. 2A). The expression of all other cytoprotective proteins assayed, as well as the pro-apoptotic protein, Bax, remained stable even after a 16-h exposure. Decreases in the protein levels of both FLIP and Mcl-1 were demonstrated within 2 h of CHX exposure, a time frame that precedes the onset of LPSϩCHX-induced apoptosis (Fig. 2B).
Interestingly, exposure of EC to both LPS and CHX resulted in a greater decrease in Mcl-1 expression at longer time points (Ն4 h) than EC exposed to CHX alone. This latter finding may suggest that, after activation of EC apoptosis by LPS, Mcl-1 becomes a downstream target of caspases. In contrast, diminished levels of FLIP were comparable in EC exposed to CHX alone or in combination with LPS.
FLIP is an anti-apoptotic protein with significant homology to caspase-8 (47). A substitution of two amino acids in the region of FLIP that corresponds to the catalytic active site of caspase-8 renders it incapable of proteolysis. The role of FLIP in the inhibition of FAS death signaling has been well elucidated. Upon Fas ligand binding, the adapter protein FADD is recruited to the Fas receptor via an interaction between the DD of each protein (48). FADD, in turn, can recruit FLIP and pro-caspase-8 via protein-protein interactions of death effector domains contained within all three proteins. Pro-caspase-8 has intrinsically low levels of proteolytic activity, which enables it to cleave other pro-caspase-8 proteins brought into close proximity following their recruitment to FADD (49). Activation of caspase-8 initiates a proteolytic cascade resulting in the activation of downstream effector caspases (50). FLIP has been proposed to inhibit activation of upstream initiator caspases, including caspase-8, by competitively binding to FADD and blocking assembly of a functional death signaling complex (51). Alternatively, FADD recruitment of both pro-caspase-8 and FLIP may occur; however, the lack of intrinsic FLIP proteolytic activity prevents the transactivation of pro-caspase-8 (34).
The TLR family of receptors has been implicated in LPS signaling (25,52). An adapter protein that plays an integral role in TLR signaling, MyD88, contains a conserved DD (28). We have previously reported that another DD-containing protein, FADD, is required for LPSϩCHX-induced apoptosis (11). A recent report suggests that MyD88 can interact with FADD through reciprocal binding of their DD (53). Analogous to the Fas pathway, the involvement of FADD in the LPS death pathway is compatible with a protective role for FLIP.
Mcl-1 is an anti-apoptotic member of the Bcl-2 family of proteins primarily localized to mitochondrial membranes (54). The Bcl-2 family contains both pro-and anti-apoptotic members capable of heterodimerization. In response to certain apoptotic stimuli, the relative ratios of these pro-and antiapoptotic proteins can dictate whether a cell will survive or undergo apoptosis (55). Bcl-2 exerts its protection by preventing the release of cytochrome c from the mitochondrion, the latter being a co-factor with Apaf-1 in the activation of procaspase-9 (56,57). Activation of caspase-9 results in a cascade effect on the activation of effector caspases culminating in apoptosis. Reportedly, Bcl-2 can also block killing downstream of and independent of cytochrome c release (58).
Mcl-1, unlike other Bcl-2 members, contains two PEST sequences, which target it for rapid turnover via proteasomemediated degradation (54). Correspondingly, inhibition of new protein synthesis resulted in decreased expression of Mcl-1, but no change in the expression levels of other Bcl-2 family members, including Bcl-2, Bcl-x L , and Bax ( Fig. 2A). The implication of a cytoprotective role for Mcl-1 is consistent with previous reports that overexpression of other Bcl-2 homologues, including Bcl-x L and A1, confers partial protection against LPS-or TNF-␣-induced EC apoptosis (30,31). An essential protective role, however, for Bcl-x L and A1 at physiological levels is uncertain as selective inhibition of their expression by antisense oligonucleotides fails to sensitize EC to TNF-␣-mediated killing (32).

Caspase Inhibition Blocks LPS-induced Apoptosis but Fails to Protect against Decreased FLIP and Mcl-1 Expression-The
cell-permeable peptide, z-VAD, a highly specific caspase inhibitor with broad selectivity for several members of the caspase family (59, 60), completely protected against LPSϩCHX-induced EC apoptosis (Fig. 3, A and B). Caspase inhibition blocked decreased EC viability (Fig. 3A), cleavage of the caspase substrate, PARP (Fig. 3B), and histone release (data not shown) in response to LPSϩCHX exposure. These findings are in agreement with previous reports that LPSϩCHX-induced EC death is apoptotic in nature and mediated through caspase activation (11).
During LPS-induced EC apoptosis, cellular proteins are cleaved after caspase activation (10). To determine whether the decreased levels of FLIP and Mcl-1 could be attributed to caspase-mediated proteolytic degradation, EC were exposed to LPSϩCHX in the presence or absence of z-VAD (Fig. 3C). Caspase inhibition did not block decreased FLIP expression and offered only modest protection against changes in Mcl-1 levels. This latter observation is consistent with the findings in Fig. 2 that the decrement in Mcl-1 expression following CHX exposure is enhanced with the co-administration of LPS, suggesting that Mcl-1 is also a substrate for activated caspases. The caspase-independent decrease of FLIP and Mcl-1 is consistent with the decrease in their expression preceding both cleavage of PARP, a known caspase substrate, and the onset of apoptosis. Furthermore, CHX exposure alone decreased the expression levels of both FLIP and Mcl-1 in the absence of detectable apoptosis. These data suggest that decreased EC levels of FLIP and Mcl-1 following CHX or LPSϩCHX exposure are due to intrinsically short half-lives of these proteins and not apoptotic processes.
Proteasome Inhibition Protects against FLIP and Mcl-1 Degradation and LPSϩCHX-induced EC Apoptosis-There are two predominant mechanisms for intracellular degradation of proteins, one involving the lysosomal apparatus and the other involving the proteasome (61). The lysosomal pathway is primarily involved in the proteolytic degradation of membranebound proteins and extracellular proteins, whereas the protea- some pathway is primarily responsible for degradation of cytosolic proteins. To determine whether CHX-induced decreases in the levels of FLIP and Mcl-1 could be attributed to rapid turnover via the proteasome, EC were pre-treated with a highly specific proteasome inhibitor, lactacystin, and its derivative ␤-lactone (62,63), and subsequently exposed to medium, LPS, CHX, or LPSϩCHX (Fig. 4A). Lactacystin protected against decreased levels of both FLIP and Mcl-1 in the presence of CHX, indicating that, once protein synthesis is inhibited with CHX, FLIP and Mcl-1 levels are rapidly decreased through a degradation process involving the proteasome.
Based upon the findings that 1) decreased levels of FLIP and Mcl-1 occur independently of caspase activation, 2) changes in FLIP and Mcl-1 expression precede the onset of apoptosis, and 3) inhibition of new protein synthesis is required for EC sensitization to LPS, a consequence of which is decreased expression of these two cytoprotective proteins, we hypothesized that preventing FLIP and/or Mcl-1 degradation would block LPSϩCHX-induced apoptosis. Therefore, EC were pre-treated with lactacystin and ␤-lactone, exposed to medium, LPS, CHX, or LPSϩCHX, and viability assessed (Fig. 4B). Under identical experimental conditions, proteasome inhibition, which prevented the degradation of FLIP and Mcl-1, also conferred complete protection against LPSϩCHX-induced decrements in viability. Measurement of histone release confirmed a protective effect of proteasome inhi-bition against LPSϩCHX-induced apoptosis (data not shown).
The requisite inhibition of new gene expression necessary for EC sensitization to LPS-induced apoptosis suggests that either a constitutive or inducible protein is required for cytoprotection. That inhibition of proteasome-dependent protein degradation in the absence of protein synthesis blocks LPSϩCHXevoked apoptosis implicates a cytoprotective role for a constitutively expressed protein(s). In contrast to this finding, a previous study reports that overexpression of an inducible protein, A1, partially protects against LPSϩCHX-and TNF-␣ϩCHX-induced EC apoptosis (30,31). Since the authors report that overexpression of two other cytoprotective proteins, Bcl-x L and A20, also confer partial protection, there is some question as to the physiological implications of these overexpression data. In fact, selective inhibition of A1 gene expression fails to sensitize EC to direct TNF-␣ killing, suggesting EC sensitization following CHX exposure is not attributed to the inhibition of A1 induction (32).
Proteasome Inhibition Confers Protection against Apoptosis Elicited by TNF-␣ϩCHX or Il-1␤ϩCHX-TNF-␣ and Il-1␤ are established mediators of the acute phase response to Gramnegative bacterial infections (64 -66). Similar to LPS, their ability to induce EC apoptosis requires the inhibition of new gene expression (Fig. 5A). To determine whether a common cytoprotective pathway is operative in the resistance of EC to these inflammatory mediators, EC were pre-treated with lactacystin and subsequently exposed to either TNF-␣ or Il-1␤ in the presence of CHX (Fig. 5B). Lactacystin completely protected against apoptosis elicited by these agents. Interestingly, lactacystin conferred only marginal protection against apoptosis elicited by staurosporine (Fig. 5B), which evokes direct EC killing without requisite inhibition of new gene expression (Fig. 5A).
A common signaling pathway shared by LPS, TNF-␣, and IL-1␤ involves the activation of the transcription factor, NF-B (67). NF-B regulates the expression of several gene products involved in inflammatory responses, including up-regulation of TNF-␣, Il-1␤, IL-6, IL-8, VCAM-1, and E-selectin (67,68). In unstimulated cells, NF-B is sequestered in the cytoplasm by members of the IB family. Upon cell activation, IB is phosphorylated on two serine residues, targeting it for proteasomemediated degradation. The resultant free NF-B is then able to translocate to the nucleus, where it promotes new gene expression.
Lactacystin, by virtue of its ability to inhibit proteasome activity, should block NF-B activation by blocking the degradation of its inhibitor, IB. As predicted, lactacystin completely blocked LPS-, TNF-␣-, and IL-1␤-induced up-regulation of VCAM-1, the expression of which is dependent on NF-B activation (Fig. 6A). These data are in agreement with a previous report that lactacystin inhibits EC NF-B activation assayed by an electrophoretic mobility shift (69).
The role of NF-B in apoptosis has been predominantly antiapoptotic through its ability to up-regulate cytoprotective gene products, including cIAPs (70,71). Recently, however, a requisite role for NF-B in promoting apoptosis has been reported (72). Therefore, it was important to rule out that lactacystin was protecting against LPSϩCHX-induced apoptosis by blocking NF-B activation through inhibition of proteasome-mediated degradation of the NF-B inhibitor, IB. Using an adenovirus transduction system, EC were transduced with either a gene for ␤-galactosidase or IB␣M with serine to alanine mutations at residues 32 and 36 (14). These serine to alanine mutations prevent signal-induced phosphorylation and proteasome-mediated degradation of the IB␣M protein (14,40). LPS-, TNF-␣, or IL-1␤ each up-regulated VCAM-1 expression in non-transduced EC and EC transduced with a control gene, but not in those transduced with the IB␣M (Fig. 6B). In contrast, IB␣M transduction did not affect the sensitivity of EC to LPS-, TNF-␣-, or IL-1␤-induced killing at 12 h in the absence of de novo protein synthesis (Fig. 6C). These data suggest that the protective effect elicited by lactacystin is through inhibition of the degradation of a cytoprotective protein, and not through the blockade of a pro-apoptotic NF-B signaling pathway. Further, the presumed mechanism of the pro-apoptotic properties of NF-B reported by Ryan et al. (72) is through the promotion of new gene expression, similar to its anti-apoptotic role and the induction of cIAPs. The finding that LPS, TNF-␣, and IL-1␤ all stimulate EC apoptosis in the absence of new protein synthesis argues against a pro-apoptotic role for NF-B involving new gene expression. Finally, transduction of EC with the IB␣M gene sensitizes these cells to apoptosis induced by long term exposure (Ն24 h) to TNF-␣, implicating an anti-apoptotic role for NF-B and not a proapoptotic role (14).
FLIP Antisense Sensitizes EC to LPS-induced Apoptosis at Low CHX Concentration-To elucidate a definitive anti-apoptotic role for either FLIP and/or Mcl-1 against LPS-induced apoptosis, antisense oligonucleotides were designed to specifically reduce the expression of each protein. Western blot analysis of EC transfected with either FLIP or Mcl-1 antisense revealed a dramatic decrement in the expression of FLIP and Mcl-1, respectively, compared with EC transfected with mismatch oligonucleotide controls (Fig. 7, A and C). Under identical conditions, we exposed the transfected cells to medium or LPS (100 ng/ml) for 12 h and assayed for histone release (Fig.  7, B and D). The decrease in FLIP or Mcl-1 with antisense oligonucleotides failed to directly sensitize EC to LPS-induced apoptosis. Since the antisense oligonucleotides reduced the level of each protein but failed to completely ablate expression, we postulated that minimal threshold level expression of either protein could confer protection. To test this hypothesis, we exposed EC transfected with FLIP or Mcl-1 antisense to LPS and a low concentration of CHX (2 g/ml) and assayed for protein expression levels (Fig. 7, A and C) and apoptosis (Fig. 7,  B and D). FLIP antisense in combination with low dose CHX (2 g/ml) resulted in almost complete knockout of FLIP expression (Fig. 7A) and sensitized EC to LPS killing (Fig. 7B). In contrast, EC transfected with mismatch control oligonucleotides had detectable levels of FLIP expression and were resistant to LPS killing. Doubling the concentration of CHX sensitized these cells to LPS killing, albeit to a lesser extent than those transfected with FLIP antisense (Fig. 7B). These data implicate a role for the constitutively expressed protein FLIP in conferring protection against LPS-induced EC apoptosis. Using EC transfected with either FLIP antisense, Mcl-1 antisense, or appropriate mismatch control oligonucleotides were exposed to LPS (100 ng/ml) in the presence or absence of CHX (2 g/ml) for 12 h. EC lysates were immunoblotted with anti-FLIP or anti-Mcl-1 antibodies (A and C). Molecular mass (in kDa) is indicated. In other experiments, EC transfected as above were exposed to a fixed concentration of LPS (100 ng/ml) in the presence of increasing concentrations of CHX for 12 h, and assayed for cytosolic histone release (B and D). Mean (Ϯ S.E.) histone release is reported in OD units. *, significantly increased compared with simultaneous mismatch oligonucleotide control.
FIG. 8. Schematic diagram of a constitutive cytoprotective pathway that protects EC from inflammatory mediator-induced apoptosis. Under physiological conditions, constitutive expression of a cytoprotective protein(s) confers resistance to LPS-, TNF-␣-, and IL-1␤induced apoptosis in human EC. Inhibition of de novo protein synthesis with CHX sensitizes EC to apoptosis elicited by each of these inflammatory mediators. In the absence of new protein synthesis, pre-existing levels of a cytoprotective protein(s) are rapidly diminished via proteasome-mediated degradation. Decreased levels of this cytoprotective protein(s) enable LPS-, TNF-␣-, or IL-1␤-induced caspase activation resulting in EC apoptosis. Proteasome inhibition with lactacystin blocks the degradation of the cytoprotective protein(s), thus, preventing CHX sensitization of EC to inflammatory mediator-induced apoptosis. In addition to a constitutive cytoprotective pathway, an inducible pathway dependent upon NF-B activation has been reported which confers human EC resistance to TNF-␣-, but not LPS or IL-1␤-induced apoptosis. a low dose of CHX in combination with FLIP antisense, cellular levels of FLIP were reduced to barely detectable limits, and sensitivity to LPS killing was conferred. Importantly, this concentration of CHX alone failed to sensitize control EC to LPS killing, indicating that the specific reduction of FLIP sensitized EC to LPS-induced apoptosis. FLIP levels in control-transfected EC exposed to low dose CHX (2 g/ml) were comparable to those in EC transfected with FLIP antisense without CHX exposure (Fig. 7A). In both cases, sensitivity to LPS killing was not observed, suggesting that a threshold level of FLIP conferred cytoprotection. At higher concentrations of CHX, a dosedependent increase in apoptosis among control-transfected EC was demonstrated (Fig. 7B), consistent with Fig. 2, in which high concentrations of CHX completely diminished FLIP levels. Although this finding clearly implicates a cytoprotective role for FLIP, it does not preclude the requisite involvement of another unidentified cytoprotective protein. It is possible that sensitivity to LPS killing was conferred to EC transfected with FLIP antisense, not only by reducing FLIP expression below minimal threshold levels necessary for protection, but also by reducing the level of another cytoprotective protein.
Similar to FLIP antisense, Mcl-1 antisense in the presence of low dose CHX (2 g/ml) decreased Mcl-1 expression below detectable levels (Fig. 7C). However, in contrast to FLIP antisense, Mcl-1 antisense neither sensitized EC to LPS-induced apoptosis nor enhanced killing compared with mismatch oligonucleotides in the presence of any concentration of CHX tested (Fig. 7D).
In summary, we have characterized a cytoprotective pathway in EC that confers resistance against LPS-induced apoptosis (Fig. 8). First, we established that inhibition of gene expression sensitizes EC to LPS-evoked killing by blocking de novo synthesis of a constitutively expressed cytoprotective protein(s), not an inducible protein. Second, this cytoprotective protein(s) has a relatively short half-life and is degraded through the proteasome. Third, EC apoptosis induced by two other inflammatory mediators, TNF-␣ and IL-1␤, in combination with a protein synthesis inhibitor is blocked by proteasome inhibition, suggesting a common cytoprotective pathway dependent upon constitutive expression of an anti-apoptotic protein(s). Fourth, by screening EC for known constitutively expressed anti-apoptotic proteins, the expression of which is acutely sensitive to inhibition of new gene expression and proteasome degradation, we identified a potential cytoprotective role against LPS-induced apoptosis for two proteins, FLIP and Mcl-1. Finally, using antisense oligonucleotides, we have identified a definitive role for FLIP in conferring protection against LPS-induced apoptosis in human EC.