From the Division of Research Technologies, Lilly Research Laboratories, Lilly Corporate Center, Indianapolis, Indiana 46285
Received for publication, January 12, 2001, and in revised form, February 1, 2001
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ABSTRACT |
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Human protein C is a natural
anticoagulant factor, and a recombinant activated form of the molecule
(rhAPC) is completing clinical evaluation for treatment of severe
sepsis. Because of the pathophysiologic role of endothelial dysfunction
in severe inflammatory disease and sepsis, we explored the possibility
that rhAPC might directly modulate endothelial function, independent of
its anticoagulant activity. Using broad transcriptional profiling, we
show that rhAPC directly modulates patterns of endothelial cell gene
expression clustering into anti-inflammatory and cell survival
pathways. rhAPC directly suppressed expression of p50 and p52 NF Endothelial dysfunction plays a critical role in uncontrolled
inflammatory conditions such as sepsis and multiorgan dysfunction syndrome. The elucidation of cytokines following insult, particularly tumor necrosis factor Human protein C is a plasma serine protease that plays a key role in
maintaining normal hemostasis (5, 6). The thrombin-activated form of
protein C (APC) acts as a feedback inhibitor of the coagulation cascade
and has demonstrated antithrombotic activity in numerous model systems
(7). APC has shown efficacy in models of lethal endotoxemia and has
been reported to prevent microvascular coagulation in patients with
meningococcal sepsis (8-12). A recombinant version of human activated
protein C (rhAPC) is completing clinical evaluation in patients with
severe sepsis, targeting the effects of microvascular coagulation.
However, there has been considerable speculation both historically and
recently (13) as to the mechanism of the apparent efficacy of APC in
the treatment of sepsis, with the implication that the beneficial
activity must be more that just antithrombotic.
Although the critical role of APC in modulating microvascular
coagulation through the inhibition of thrombin generation has been well
studied, its direct effects on endothelial function have not been
elucidated. We used a novel approach of broad transcriptional profiling
and cluster analysis to define possible new molecular mechanisms for
the APC pathway in vascular endothelial function. Transcript profiling,
the simultaneous monitoring of gene expression for a significant
portion of a genome, has emerged as a powerful tool in genetics and
biology, because it allows for the analysis of many of the signal
transduction pathways and other biological systems identified from the
sequencing of the human genome (14). This profiling has been used to
reveal novel targets and mechanisms of action using yeast as a model
system (15-17). Using this novel approach, we demonstrate that rhAPC
directly modulates cell signaling and alters gene expression in two
major pathways of inflammation and apoptosis. rhAPC suppressed
NF Cell Culture--
Human umbilical vein endothelial cells
(HUVEC) and pools HUVEC PO96, P146, or P150 were grown in
endothelial growth medium with 2% FBS (Clonetics, San Diego,
CA). Cells were prepared untreated or treated with rhAPC (Eli
Lilly and Co., Indianapolis, IN) and/or TNF Microarray--
Cells were washed, and total RNA was isolated by
TriazolTM (Life Technologies, Inc.) following the
manufacturer's recommendations. RNA was stored at RT-PCR--
A semiquantitative RT-PCR assay was used to follow
up on Affymetrix data. First-strand cDNA was synthesized from 4 µg of total RNA (Superscript; Life Technologies, Inc.). After
first-strand synthesis, the cDNA was diluted to a final volume of
160 µl and normalized using DNA Binding Assay--
Reactions containing Buffer D (20 mM HEPES, pH 7.9, 100 mM KC1, 5 mM
MgC12, 0.2 mM EDTA, 0.5 mM
dithiothreitol), poly(dI·dC), nuclear extract, and labeled
oligonucleotide were incubated for 15-45 min. In some experiments
antibodies to NF Flow Cytometry--
Primary antibody at 1-2 µg/ml in 100 µl
of FACS buffer (PBS, 5% albumin, 0.02% sodium azide) was applied at
4 °C for 30 min. The secondary antibody, anti-mouse IgG-FITC, at 1 µg/ml in 100 µl of FACS buffer was applied at 4 °C for 30 min.
FACS analysis was done with a CoulterTM flow cytometer
(Coulter). Primary antibodies were to adhesion markers ICAM-1,
E-selectin, VCAM-1, and fractalkine (R & D Systems, Minneapolis, MN).
Apoptosis Assay--
Cells were seeded at 3 × 104 cells per well in a 96-well plate and treated with 1 µg/ml staurosporine (Sigma) for 1 h or with staurosporine and
APC (pretreatment for 16 h). Cells were prepared and stained
according to APOPercentageTM apoptosis assay per the
manufacturer's instructions (Biocolor Ltd., Belfast, Northern Ireland).
Transcript Profile of rhAPC Alone and in TNF-treated
Endothelial Cells--
The effect of rhAPC on TNF-activated or
non-activated human endothelial cells was assessed using transcript
profiling with AffymetrixTM microarrays. Treatments were
performed under conditions where no thrombin or other protease activity
could be detected, and no detectable thrombin was being generated
during the cell culture experiments as described under "Experimental
Procedures." After treatment, cells were lysed and mRNA was
isolated, followed by analysis on AffymetrixTM arrays, and
results were confirmed by semiquantitative RT-PCR. We found that rhAPC
and TNF treatments of HUVECs resulted in 10 genes that were regulated
by rhAPC alone and 31 genes regulated by TNF alone. Shown in
Table I are the genes whose expression was altered by rhAPC treatment alone and 13 genes of the 31 TNF-regulated genes that we found to be comodulated by rhAPC. In
general, the genes clustered with respect to cellular function into
those involved in inflammation/immune modulation and cell
survival/apoptosis. We also observed that the TNF-activated genes,
which primarily fell into the proinflammatory and apoptotic pathways,
were counter modulated by the rhAPC treatment. There were several
TNF-activated genes, associated with feedback pathways for controlling
TNF responses, (19) that were further enhanced by rhAPC treatment.
Notably, the TNF-activated A20 gene, which was enhanced by rhAPC, has
recently been shown in knockout experiments to be critical in
regulating TNF-induced effects and cell death responses (20). Overall, the pattern of gene expression modulated by rhAPC was consistent with
an induction of anti-inflammatory and antiapoptotic pathways.
Effect of rhAPC on the NF Suppression of Cell Adhesion Molecules by rhAPC--
As
shown in Table I, a number of TNF-modulated genes were
counter-modulated by rhAPC, and the induction of many of these genes
has previously been shown to be mediated through NF APC-dependent Modulation of Apoptosis--
In addition
to the described effect of rhAPC on NF
Considering this pattern of gene expression, experiments were performed
to determine whether rhAPC could actually block the induction of
apoptosis. As shown in Fig. 3B, Eahy926 endothelial cells
treated with staurosporine, a potent inducer of the cell death pathway,
underwent apoptotic cell death by 3 h post-treatment. However,
treatment with rhAPC significantly reduced the number of cells
undergoing apoptosis. We observed a similar protective effect using
kidney 293 cells and HUVECs, which were extremely sensitive to
apoptotic induction. In repeated and quantified experiments, rhAPC
significantly blocked the induction of apoptosis in each of these cells
(Fig. 3C). The effect of rhAPC on the inhibition of
apoptosis was dose-dependent with concentrations as low as ~2 nM effectively suppressing the induction of apoptosis.
Further, these apoptosis results were confirmed by intracellular
staining of caspase 3 (data not shown).
As indicated above, we observed no significant thrombin
generation during the course of our cell culture experiments analyzing the effect of APC on endothelial function. However, to assure that the
observed effects were independent of the ability of APC to inhibit the
generation of thrombin, studies were conducted in the presence of
hirudin, a potent thrombin inhibitor. In repeated experiments in which
hirudin was included in the cell culture treatments, we found no effect
of this inhibitor on the anti-inflammatory and apoptosis responses with
or without APC (data not shown). In the apoptosis analysis shown in
Fig. 3, the addition of hirudin had no effect on the ability of APC to
suppress staurosporine-induced apoptosis. The addition of hirudin also
had no effect on the apoptosis suppression by APC as measured by
caspase 3 staining. Moreover, we found no effect of thrombin inhibition
on the ability of APC to suppress the TNF activation of the cell
surface adhesion markers as in Fig. 2, measuring both VCAM and ICAM
levels. Thus, the effect of APC on modulating endothelial cell pathways
was independent of its ability to inhibit thrombin generation.
We have utilized a novel approach to define new molecular pathways
for the subcellular action of the anticoagulant human protein C. Moreover, our data demonstrate the power of transcriptional profiling
in defining drug mechanism and implications for disease therapy. In
Fig. 4, we diagram the expanded paradigm
of the physiological role of APC. We have found that beyond its
indirect effect on inflammation, via inhibition of thrombin generation,
APC has novel direct anti-inflammatory effects via suppression of the
NFB
subunits, resulting in a functional decrease in NF
B binding at
target sites. Further, rhAPC blocked expression of downstream NF
B
regulated genes following tumor necrosis factor
induction, including dose-dependent suppression of cell
adhesion expression and functional binding of intracellular adhesion
molecule 1, vascular cell adhesion molecule 1, and E-selectin. Further, rhAPC modulated several genes in the endothelial apoptosis pathway, including the Bcl-2 homologue protein and inhibitor of apoptosis protein. These pathway changes resulted in the ability of rhAPC to inhibit the induction of apoptosis by the potent inducer,
staurosporine. This new mechanistic understanding of endothelial
regulation and the modulation of tumor necrosis factor-induced
endothelial dysfunction creates a novel link between coagulation,
inflammation, and cell death and provides insight into the molecular
basis for the efficacy of APC in systemic inflammation and sepsis.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(TNF),1 initiates cell
surface activation affecting a number of pathways (e.g. oxidation, adhesion, cytokine release,
apoptosis, and nitric oxide production), as well as releasing a tissue
factor that further contributes to inflammation though thrombin-induced
activation of endothelium, platelets, and vascular smooth muscle (1). The overall result of this cellular activation is a dysregulation of
endothelial function, leading to microvascular thrombosis, end organ
damage, multiple organ dysfunction, and often death (2, 3). In the last
several years, blocking disseminated intravascular coagulation, and the
microthrombi that may promote end-organ dysfunction, has been proposed
as a new target for clinical treatment in sepsis (4).
B-modulated genes by directly reducing NF
B expression and
functional activity. Further, rhAPC inhibited cytokine signaling,
including TNF-induction of cell surface adhesion molecules
(e.g. VCAM, ICAM, E-selectin, and fractalkine). rhAPC also modulated apoptosis pathways, including up-regulation of the
endothelial Bcl-2 homolog (A1), eNOS, and the inhibitor of apoptosis
(IAP), and suppression of the apoptosis-associated genes calreticulin
and TRMP-2. Moreover, treatment of cells with rhAPC blocked the
induction of apoptosis. These data provide a novel view of how
inflammation is modulated at the endothelial cell level and defines new
relationships among hemostasis, inflammation, and cell death/apoptosis.
Moreover, our results demonstrate the value of gene profiling in
defining novel mechanisms of drug action and specifically in providing
possible mechanistic answers to rhAPC efficacy in sepsis.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(R & D Systems,
Minneapolis, MN). For the AffymetrixTM experiment, cells were treated
with APC for 16 h and TNF for the last 7 h. For the four
conditions (untreated; vehicle of 0.1% bovine serum albumin in
Tris/NaCl, 183 nM APC; 1 ng/ml TNF; and 1 ng/ml
TNF plus 183 nM APC) cells were observed for a total of
16 h. EAhy926 cells (17), obtained from the University of North
Carolina, were grown in T-75 flasks with Dulbecco's modified Eagle's
medium/F-12 (3:1), 5% FBS, 50 µM/liter gentamicin, and
20 mM/liter HEPES. Cells were grown to confluence
(70-90%) and were treated under various conditions as described in
individual experiments. TNF concentrations were 1 ng/ml, and APC ranged
from 8 nM to 180 nM. Cells were treated at
various time points between 4 and 48 h. In control experiments,
recombinant hirudin (lepirudin rDNA) was added to the cell culture
medium at ~1 µg/ml. There was no residual thrombin in any of
the preparations as determined by amidolytic assay using S-2366
(Chromogenix) for thrombin proteolytic activity, compared with control
thrombin at subnanomolar concentrations. During the course of the
experiments, samples were tested for amidolytic activity in the
presence and absence of hirudin, and no significant thrombin activity
could be detected (<25 pM limit of detection).
80 °C in
diethyl pyrocarbonate-treated deionized water. Detailed methods
for labeling the samples and subsequent hybridization to the arrays are
available from Affymetrix (Santa Clara, CA). Briefly, 1.5 µg
of poly(A)+ RNA was converted to double-stranded
cDNA (Superscript; Life Technologies, Inc.) priming the
first-strand synthesis with a T7-(dT)24 primer containing a
T7 polymerase promoter. 1 µg of double-stranded cDNA was
subsequently used as a template to generate biotinylated cRNA using the
incorporated T7 promoter sequence in an in vitro
transcription system (Megascript kit; Ambion and Bio-11-CTP and
Bio-16-UTP; Enzo). Control oligonucleotides and spikes were added to 15 µg of cRNA, which was then hybridized to Hu6800 oligonucleotide
arrays for 16 h at 45 °C with constant rotation. The arrays
were then washed and stained on an Affymetrix fluidics station using
the EUKGE-WS1 protocol and scanned on an Affymetrix GeneArray scanner.
Data analysis was performed using GeneChip 3.1 software. Internal
controls of housekeeping genes and a test chip trial were run prior to
test samples.
-actin as the reference. Control
primers for human cytoplasmic
-actin were as follows: primer L1, 5'
CGTCATACTCCTGCTTGCTGATCCACATCTGC 3'; and primer R1, 5'
ATCTGGCACCACACCTTCTACAATGAGCTGCG-3'. Control primers for human
transferrin receptor were as follows: primer 5pri,
5'-CTTTCTGTTTTTGCGAGGACACA-3'; and primer 3pri,
5'-TCCAAGTAGCTAGAGCCAACTGGTT-3'. Multiplexed PCR reactions were
performed containing 0.5 pmol of each actin, 7.5 pmol of each
transferrin receptor, and 5.0 pmol of each gene-specific primer. A
25-µl reaction containing 4 µl of normalized cDNA was performed
using cDNA advantage polymerase mix (CLONTECH)
using the following cycling reactions: step 1, 94 °C for 5 min; step
2, 94 °C for 45 s; step 3, 60 °C for 45 s; step 4, 72 °C for 2 min; step 5, repeat steps 2-4 19, 24, or 29 times; and
step 6, 72 °C for 7 min. 4 µl of load dye was then added
(Bluejuice; Life Technologies, Inc.), and 5 µl were loaded onto 2%
agarose gels.
B subunits were also included in the
reaction. Complexes were run on 5% (0.5× TBE) polyacrylamide gels
(Owl, Inc.). The probe was
-32P-labeled (PerkinElmer
Life Sciences), and nuclear extracts from Eahy926 and
HUVEC were prepared as described (18). Samples were run for 90 min at
110 V, 2.0 A in 1/2× TBE running buffer. Gels were dried on DE81 paper
and developed overnight on Kodak film (Eastman Kodak Co., Rochester,
NY), and monoclonal antibodies to NF
Bp65, NF
Bp50, and NF
Bp52
(NF
B2) (Santa Cruz Biotechnology, Santa Cruz, CA) were used for
specificity controls. Quantification of gel shift to a consensus
NF
B-C 5'-CAGTTGAGGGGACTCCAGGCC-3" site was performed by a
scanning phosphorimager.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Selected genes modulated by rhAPC in human endothelial cells
B-regulated are noted (N). Only genes
showing at least a 2-fold change in array experiments are listed. DEC,
decrease; INC, increase; reg., regulation.
B Pathway--
A very notable change
observed on the transcript profile following rhAPC treatment was a
suppression of p52 (NF
B2 subunit) on the array. Further analysis of
the array results indicated that expression of this NF
B subunit was
suppressed both by rhAPC alone and in combination with TNF (Fig.
1A). The average difference from the 20 perfect and mismatch signals on the Affymetrix array demonstrated a reduction in NF
B2 mRNA by rhAPC, with or without TNF cotreatment. Semiquantitative RT-PCR confirmed that the NF
B2 mRNA was suppressed by rhAPC alone and that rhAPC attenuated its induction by TNF (data not shown). To further examine the functional effect of the suppression at the mRNA level, an electrophoretic mobility shift assay (EMSA) for NF
B was used to determine the amount
of NF
B DNA binding in nuclear extracts from treated HUVECs using a
32P-labeled oligonucleotide consensus probe to NF
B.
(Fig. 1B). In repeated experiments, rhAPC alone suppressed
NF
B nuclear extract DNA binding by ~60% (n = 4),
consistent with the reduced mRNA expression (lanes 1 and
2). Moreover, in repeated experiments, rhAPC also attenuated
the amount of binding following TNF induction of NF
B (lanes
3-5). In addition to these results using HUVECs, we obtained the
same results using the Eahy926 (21) human endothelial line (data not
shown). The specificity of the EMSA binding assays was demonstrated
with antibody supershifts to p65, p50 (NF
B1), and p52 (NF
B2), as
well as competition with cold consensus sequence (data not shown). This
showed that the effect was specific and that rhAPC a can directly
inhibit the functional NF
B pathway (both NF
B1 and NF
B2) in
endothelial cells.
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Fig. 1.
APC down-regulates
NF B gene expression in HUVECs.
A, effect of rhAPC on mRNA levels by Affymetrix analysis
and semiquantitative RT-PCR. Levels of NF
B2 expression were compared
with untreated control cells. Data represents the average difference
from 20 perfect and mismatch signals on the Affymetrix chip (duplicate
chips and confirmed by RT-PCR at two cycle times with actin and
transferrin receptor controls). B, functional analysis of
NF
B activity using a DNA shift assay (mean ± S.D.;
n = 5). Gel shows functional suppression of NF
B DNA
consensus sequence binding when excess radiolabeled (32P)
consensus sequence is present. Lane 1, nuclear extract from
untreated cells; lane 2, with rhAPC alone (320 nM, 24-h pretreatment); lane 3, nuclear extract
from untreated cells; lane 4, cells treated with TNF alone
(1 ng/ml for 4 h); and lane 5, rhAPC pretreatment
followed by TNF.
B (see Table I).
We focused additional studies on the adhesion molecules as being of
particular importance with regard to anti-inflammatory effects.
Semiquantitative RT-PCR confirmed that rhAPC mediated suppression of
several of the adhesion molecule mRNAs (induced by TNF) including
CX3C (fractalkine) (22), ICAM-1, E-selectin, and VCAM-1 (23). By flow
cytometry, we confirmed that ICAM-1 surface expression induced by TNF
could be suppressed by rhAPC in endothelial cells (Fig.
2A). The effect of APC on the
reduction in ICAM-1 expression was concentration-dependent,
as shown in Fig. 2B. Similarly, we have shown that rhAPC
could inhibit the expression both E-selectin and VCAM-1, two other
important leukocyte adhesion molecules. As shown in Fig. 2C,
rhAPC reduced cell surface E-selectin expression, measured by flow
cytometry, in a concentration-dependent manner. Further,
using a direct cell binding assay, dependent on the interaction of
endothelial VCAM-1 and very late antigen-4 on a target cell
(U937), we demonstrate that rhAPC directly inhibits the cell-cell
interaction up-regulated by TNF (Fig. 2D). Additional studies with VCAM-1 and fractalkine showed comparable
dose-dependent inhibition by rhAPC.
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Fig. 2.
rhAPC suppresses
NF B-activated surface adhesion proteins ICAM-1
and E-selectin. A, analysis of ICAM-1 surface
expression by flow cytometry in human endothelial cells. Cells were
treated with rhAPC, TNF, or both combined, and the levels of mean
fluorescence intensity versus events was determined using an
FITC-labeled secondary antibody as described below. B, dose
response for the effect of rhAPC on TNF-induced ICAM surface expression
(mean ± S.D.; n = 3). C, dose response
for the effect of rhAPC on TNF-induced E-selectin surface expression
similarly determined by flow cytometry. In both B and
C the levels were made relative to untreated control as
100%. D, effect of rhAPC on VCAM-dependent
cell-cell interaction (mean ± S.D.; n = 5). Very
late antigen-4-expressing U937 mononuclear cells were used in
endothelial cell adhesion experiments using procedures described
previously (42).
B and its downstream mediation
of inflammation, our profiling results also suggested a second
clustering around pathways promoting antiapoptosis and cell survival.
As shown in Fig. 3A, rhAPC
suppress two proapoptotic genes, calreticulin, an endoplasmic reticulum
luminal protein that when suppressed has been shown to decrease cell
apoptosis (24), and TRMP-2, a marker of cell apoptosis (25). In
contrast, rhAPC increased genes shown to be antiapoptotic or markers of cell survival such as A1 (19), IAP (26), cell cycle-related human Gu
helicase (27), and proliferating cell nuclear antigen (PCNA) (28).
Interestingly, rhAPC also significantly up-regulated eNOS, which is
important in view of the significant role it plays as an endothelial
survival factor (reviewed in Ref. 29).
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Fig. 3.
rhAPC modulates genes associated with cell
survival and apoptosis. A, selected gene changes
affected by rhAPC related to cell cycle and apoptosis. The results are
depicted as -fold differences determined from the average difference
change from Affymetrix arrays (see "Experimental Procedures") and
made relative to actin controls (data from duplicate array experiments
each with 20 perfect and mismatch signals on the array). B,
protective effect of rhAPC against induction of apoptosis. Effect of
rhAPC treatment (8 nM) on staurosporine (SS; 1 µM)-induced apoptosis in human endothelial cells and
kidney 293 cells is shown. C, quantification of
staurosporine-induced apoptosis in Eahy926, kidney 293 cells, and
HUVECs with and without rhAPC (mean ± S.D.; n = 4).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B pathway and an apparent ability to prevent apoptosis and
modulate cell survival. This effect of rhAPC on inhibiting apoptosis
appears to be separate and antithetical to most of the TNF/NF
B
pathway effects, yet consistent with an effect that would be beneficial in antithrombotic/anti-inflammatory situations. In fact, there is
growing evidence for the role of apoptosis in systemic inflammatory response and sepsis (30-32). The emerging data suggest that
organs-specific cell death involving both parenchymal and microvascular
endothelium underlies organ dysfunction, with increased apoptotic rates
occurring in organ dysfunction. The concept of suppression of
proinflammatory pathways and the switch to cellular survival mechanisms
at the endothelial interface suggests a complex adaptive response at the vessel wall directly connected at the subcellular level to the
action of APC. The protein C pathway in inflammatory states could
protect the organism from vascular insult and possibly prolong endothelial, cellular, and organ survival.
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Fig. 4.
Model for the expanded role for protein C
pathway in endothelial cell. In addition to its classic role as a
feedback inhibitor of thrombin generation, APC suppress TNF-mediated
effects though the down-regulation of NF B subunits and subsequent
inhibition of inflammatory cell adhesion. Moreover, APC modulates
pathways associated with an antiapoptotic phenotype.
The ability of APC to suppress the up-regulation of adhesion molecules demonstrates a significant anti-inflammatory activity and, in conjunction with antithrombotic activity, further supports its role in severe systemic inflammatory disorders, including sepsis. The latter is strongly supported by studies in the ICAM-1 knockout mouse with no endothelial surface ICAM-1 that is resistant to induction of sepsis (33) and protected from ischemic renal injury (34). Recently, diminished neutrophil myeloperoxidase activity in a renal ischemia reperfusion animal model suggested suppression of inflammation related to APC and adhesion, further supporting our proposed mechanism (35). Thus, failure of endothelial function in severe inflammatory processes without the immune modulatory influences of APC may help explain both the adverse outcomes of septic patients with protein C deficiency and the favorable outcomes seen in meningococcal purpura fulminans patients receiving protein C and APC treatment (9, 12, 36).
Further investigation of the broader effects of NFB suppression by
APC on endothelium and immune leukocytes may help expand our
understanding of the link between coagulation and inflammation and
those disease states where protein C pathway regulation is important.
APC may play a physiological role in other disease states involving
endothelial inflammation and apoptosis e.g.
arteriosclerosis, acute respiratory distress syndrome, systemic
vasculitis syndromes, allograft vasculopathy (37), hepatic
veno-occlusive disease (38), and sickle cell syndromes (39). Moreover,
ongoing studies will help define the role of other possible
inflammation/immune modulation pathways suggested from our broad
profiling (e.g. chemoattractant B61 (Table I) and
the suppression of Class I human lymphocyte antigen genes; data
not shown). Recently, the endothelial protein C receptor (40) has been
demonstrated to be important in survival in the baboon sepsis model
(41). It will be important to determine whether this receptor is
involved in the cell signaling we have observed with rhAPC.
At the time of this writing, enrollment in the PROWESS trial, a phase
III, placebo-controlled, 28-day, all-cause mortality study, was stopped
early based on interim analysis showing that trial results met the
criteria for reduced mortality among rhAPC-treated patients. There has
been considerable speculation, both historically and recently (13), as
to the mechanism of the apparent efficacy of APC in the treatment of
sepsis. Our data provide additional mechanistic understanding, not only
for the physiologic role of APC, but for its potential therapeutic role
in sepsis and systemic inflammatory responses.
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ACKNOWLEDGEMENTS |
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We thank David Berg, Bruce Gerlitz, Mark Richardson, and Bryan Jones for helpful suggestions, Phil Marder and Lisa Green for expert assistance with flow cytometry, Qingqin Li for help with bioinformatic analysis, and Stacy Raper for manuscript support.
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Contributed equally to the study.
§ To whom correspondence should be addressed. Tel.: 317-276-2293; Fax: 317-277-2934; E-mail: grinnell@lilly.com.
Published, JBC Papers in Press, February 5, 2001, DOI 10.1074/jbc.C100017200
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ABBREVIATIONS |
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The abbreviations used are:
TNF, tumor
necrosis factor;
APC, activated protein C;
rhAPC, recombinant human
activated protein C;
NFB, nuclear transcription factor
B;
ICAM, intracellular adhesion molecule;
VCAM, vascular cell adhesion molecule;
A1, Bcl-2 homologue protein;
IAP, inhibitor of apoptosis protein;
eNOS, endothelial nitric oxide synthase;
HUVEC, human umbilical vein
endothelial cell;
EMSA, electrophoretic mobility shift assay;
PCNA, proliferating cell nuclear antigen;
FBS, fetal bovine serum;
PCR, polymerase chain reaction;
TBE, Tris-buffered EDTA;
FACS, fluorescence-activated cell sorter;
FITC, fluorescein
isothiocyanate.
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