3 Section of Pulmonary and Critical Care Medicine, Yale University School of Medicine, New Haven 06250; 4 Connecticut Veterans Affairs HealthCare Service, West Haven, Connecticut 06516; 2 Department of Molecular Genetics, Alton Ochsner Medical Foundation, and Department of Biochemistry and Molecular Biology, Louisiana State University Medical Center, New Orleans, Louisiana 70121; and 1 The Johns Hopkins Medical Institution, Baltimore, Maryland 21205
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ABSTRACT |
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Heme oxygenase (HO)-1
catalyzes the oxidative cleavage of heme to yield equimolar amounts of
biliverdin, iron, and carbon monoxide. HO-1 is a stress response
protein, the induction of which is associated with protection against
oxidative stress. The mechanism(s) of protection is not completely
elucidated, although it is suggested that one or more of the catalytic
by-products provide antioxidant functions either directly or
indirectly. The involvement of reactive oxygen species in apoptosis
raised the question of a possible role for HO-1 in programmed cell
death. Using the tetracycline-regulated expression system, we show here that conditional overexpression of HO-1 prevents tumor necrosis factor--induced apoptosis in murine L929 fibroblasts. Inhibition of
apoptosis was not observed in the presence of tin protoporphyrin, a
specific inhibitor of HO activity, and in cells overexpressing antisense HO-1. Interestingly, exogenous administration of a low concentration of carbon monoxide also prevented tumor necrosis factor-
-induced apoptosis in L929 fibroblasts. Inhibition of tumor
necrosis factor-
-induced apoptosis by HO-1 overexpression was
reversed by
1H-(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one, an inhibitor of guanylate cyclase, which is a target enzyme for carbon monoxide. Taken together, our data suggest that the antiapoptotic effect of HO-1 may be mediated via carbon monoxide.
tumor necrosis factor-; programmed cell death; carbon monoxide; oxidants; reactive oxygen species
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INTRODUCTION |
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APOPTOSIS IS A FORM OF CELL DEATH occurring as a result of a genetically programmed or stimulus-induced event (5, 11, 24). The role of reactive oxygen species (ROS) in the pathogenesis of apoptosis is under active investigation; it has been shown that one of the most potent antiapoptotic genes, bcl-2, has antioxidant properties (18, 32, 42). Exogenously administered ROS can trigger apoptosis in various in vitro and in vivo models, and ROS are generated intracellularly by inducers of apoptosis and appear to mediate cell death (33). Additionally, several antioxidants such as N-acetyl-L-cysteine, nigericin, copper-zinc superoxide dismutase, and catalase have been shown to inhibit apoptosis (22, 39).
Heme oxygenases (HOs) are ubiquitous enzymes that catalyze the initial and rate-limiting steps in the oxidative degradation of heme to bilirubin; using NADPH and molecular oxygen, HOs cleave a mesocarbon of the heme molecule, producing equimolar quantities of biliverdin, iron, and carbon monoxide (CO) (9). Biliverdin is reduced to bilirubin by bilirubin reductase, and the free iron is either used in intracellular metabolism or sequestered in ferritin. CO may act as a cellular messenger and has been implicated in vascular tone regulation and neurotransmission (15, 25). To date, three HO isoenzymes have been identified; of these, HO-2 and HO-3 are constitutively expressed (9, 23), whereas HO-1 is inducible (1, 9).
HO-1 is involved in the oxidative stress response (1, 7, 9), being highly induced and conferring protective effects in such conditions both in vivo and in vitro (21, 27, 28, 40). The mechanism(s) by which HO-1 confers protection against oxidative stress is not clearly understood. It is generally believed that the catalytic by-products derived from the catalysis of heme by HO, namely biliverdin, bilirubin, ferritin accumulation from released free iron, and CO, may mediate the physiological effects of HO. Both biliverdin and bilirubin possess antioxidant properties (9). Furthermore, the iron released during heme catabolism can stimulate ferritin synthesis. It has been suggested that increased levels of ferritin reduce the cellular oxidant potential by further decreasing the intracellular concentration of free iron (4, 9). A recent report has also observed that CO at low doses can provide protection against oxidant stress in vivo (29).
In light of the role of oxidative stress in apoptosis (5, 19), we
hypothesized that HO-1 may exhibit antiapoptotic activity. To date,
this hypothesis has not been tested rigorously. We used tumor necrosis
factor (TNF)--induced apoptosis of L929 mouse fibroblast cells to
test our hypothesis because besides being a well-characterized model of
programmed cell death, ROS play a critical role in mediating
TNF-
-induced apoptosis in these fibroblasts (6, 43). We used a
tetracycline-controlled expression system to induce HO-1 expression
conditionally before exposing the cells to TNF-
. We show that HO-1
induction inhibits TNF-
-induced apoptosis in L929 cells. Inducing
the antisense HO-1 or inhibiting HO-1 with tin protoporphyrin (SnPP)
reversed the HO-1-induced antiapoptotic effect. Additional experiments
were carried out to address the mechanism(s) by which HO-1 exerts its
antiapoptotic effects.
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METHODS |
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Plasmids. The tTA expression plasmid and the tTA target vector, pUHD15-1neo and pUHD10-3, respectively (12), were kindly provided by Dr. H. Bujard (Heidelberg ZMBH, Heidelberg, Germany). Plasmid pUHDluc was constructed by cloning the Photinus pyralis luciferase cDNA (10) downstream of the tetracycline operator sequences in the plasmid pUHD10-3. The rat glucocorticoid receptor expression plasmid pVARO (18) and plasmid pRHO-1 (31, 35) containing rat HO-1 cDNA were kindly provided by Drs. K. Yamamoto (University of California, San Francisco) and S. Shibahara (Tohoku University, Sendai, Japan), respectively. The hygromycin-B phosphotransferase and the puromycin-N-acetyltransferase expression plasmids pCEP4 and pPUR were purchased from Invitrogen (Carlsbad, CA) and Clontech (San Antonio, TX), respectively. All transfections were carried out with the CaPO4 precipitation technique (1).
Construction of a tetracycline-responsive HO-1 expression
plasmid. The tTA response plasmid pUHD10-3 contains seven copies of
the tetracycline (Tet) operator Tet O and the minimal human cytomegalovirus immediate-early promoter (53 to +75 bp) upstream from the multiple cloning and the SV40 small t-antigen intron and
polyadenylation sequences. This plasmid was modified by replacing the
intron and polyadenylation sequences of SV40 with analogous regions of
the rabbit b-globin gene from plasmid pVARO to yield pToG (13). The rat
HO-1 cDNA clone was digested with Xho I and Hind III,
and a 1.0-kb fragment was isolated, blunt ended, and cloned upstream
from the b-globin sequences in both the sense and antisense
orientations to yield pToG/HO-1 and pToG/OH-1, respectively.
Cell culture. E8.2.A3 cells, a glucocorticoid receptor-negative
clone of L929 cells, were used in this study and are referred to as
L929 cells. L929 cells were cultured in Dulbecco's modified Eagle's
medium containing 10% fetal bovine serum. Various antibiotics were
included in the culture medium as indicated. Cells grown in the
presence of Tet are designated L929 Tet+, whereas those cultured in
medium lacking Tet are designated L929 Tet. Recombinant murine
TNF-
was obtained from Boehringer Mannheim (Mannheim, Germany). The
dose used was 100 ng/ml unless otherwise indicated. 1H-(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one (ODQ) was
purchased from Tocris Cookson (Ballwin, MO).
RNA extraction and Northern blot analysis. Total RNA was isolated by the STAT-60 RNAzol method with direct lysis of cells in RNAzol lysis buffer followed by chloroform extraction (Tel-Test "B," Friendswood, TX). Ten micrograms of total RNA were electrophoresed on a 1% agarose gel, transferred to a nylon membrane (DuPont, Boston, MA) via capillary action, and cross-linked with an ultraviolet cross-linker (Stratagene, La Jolla, CA). The nylon membranes were prehybridized in hybridization buffer (1% BSA, 7% SDS, 0.5 M phosphate buffer, pH 7.0, and 1.0 mM EDTA) at 65°C for 2 h followed by incubation in hybridization buffer containing 32P-labeled rat HO-1 cDNA at 65°C for 24 h. Nylon membranes were then washed twice in buffer A (0.5% BSA, 5% SDS, 40 mM phosphate buffer, pH 7.0, and 1.0 mM EDTA) for 30 min at 55°C followed by four washes in buffer B (1% SDS, 40 mM phosphate buffer, pH 7.0, and 1.0 mM EDTA) for 15 min at 55°C. Ethidium bromide staining of the gel was used to confirm RNA integrity. To control for variation in either the amount of RNA in different samples or loading errors, the blots were hybridized with an oligonucleotide probe complementary to 18S rRNA after being stripped of HO-1.
cDNA and oligonucleotide probes. A full-length rat HO-1 cDNA,
generously provided by Dr. S. Shibahara was subcloned into pBluescript vector, and Hind III-EcoR I digestion was done to
isolate a 0.9-kb HO-1 cDNA insert. A 24-bp oligonucleotide
(5'-ACGGTATCTGATCGTCTTCGAACC-3') complementary to 18S rRNA
was synthesized with a DNA synthesizer (Applied Biosystems, Foster
City, CA). HO-1 cDNA was labeled with [-32P]CTP with a random-primer kit
(Boehringer Mannheim). The 18S rRNA oligonucleotide was labeled with
[
-32P]ATP at the 3'-end with terminal
deoxynucleotidyltransferase (Bethesda Research Laboratories,
Gaithersburg, MD).
Western blot analysis. For HO-1 immunoblots, the cells were homogenized in lysis buffer (1% Nonidet P-40, 20 mM Tris, pH 8.0, 137.5 mM NaCl, 1 mM Na3VO4, 1 mM phenylmethylsulfonyl fluoride, and 10 mg/ml of aprotinin). Protein concentrations of the lysate were determined by Coomassie blue dye-binding assay (Bio-Rad Laboratories, Hercules, CA). An equal volume of 2× SDS sample buffer (0.125 M Tris · HCl, pH 7.4, 4% SDS, and 20% glycerol) was added, and the samples were boiled for 5 min. Samples (100 mg) were subjected to electrophoresis in a 12% SDS-polyacrylamide gel (Novex, San Diego, CA) for 2 h at 20 mA. The proteins were then transferred electrophoretically (Bio-Rad Laboratories) onto a polyvinylidene fluoride membrane (Immobilon, Bedford, MA) and incubated for 2 h in Tris-buffered saline-1% Tween 20 buffer (TBS-T) containing 5% nonfat powdered milk. The membranes were then incubated for 2 h with a rabbit polyclonal antibody against rat HO-1 (1:1,000 dilution). The rat HO-1 antibody was purchased from StressGen (Vancouver, BC). After three washes in TBS-T for 5 min each, the membranes were incubated with goat anti-rabbit IgG antibody (Amersham, Arlington Heights, IL) for 2 h. The membranes were then washed three times in TBS-T for 5 min each followed by detection of the signal with an enhanced chemiluminescence detection kit (Amersham).
The terminal deoxynucleotidyltransferase dUTP nick end-labeling method. A two-step binding assay was used to label the 3'-hydroxyl ends of the DNA breaks, resulting in fluorescent labeling of the apoptotic cells within a population. An APO-BRDU kit (Phoenix Flow Systems, San Diego, CA) combines the terminal deoxynucleotidyltransferase enzyme to catalyze extensions of the 3'-hydroxyl ends and bromo-dUTP to substitute for thymidine (TUNEL assay). FITC-conjugated antibromodeoxyuridine results in green labeling. The cells were resuspended in RNase and propidium iodide to hydrolyze the double-stranded RNA and to label the total DNA stoichiometrically, respectively. Apoptosis was correlated with cell cycle by generating two independent fluorescent signals on the FACStarplus flow cytometer. The total DNA signal was amplified linearly, whereas the antibromodeoxyuridine signal was amplified logarithmically. The digital output was correlated as two-parameter dot plots.
Genomic DNA isolation DNA-laddering assay. Genomic DNA was isolated from cultured cells with the Puregene DNA Isolation Kit (Gentra Systems, Minneapolis, MN). Briefly, cells were lysed after medium removal with lysis buffer followed by a 1-h incubation with RNase A. The cell lysates were precipitated for proteins and spun at 2,000 g for 15 min. The supernatant was precipitated with isopropanol for isolation of DNA. After an alcohol wash, DNA was hydrated and quantified. Equal amounts (20 µg) of DNA were electrophoresed on a 1.5% agarose gel (with incorporated ethidium bromide) in 1× Tris-acetate buffer. The gel was then photographed under ultraviolet luminescence.
Nucleosomal ELISA assay. Cell lysates were isolated, and ELISA assays were performed according to manufacturer's protocol (Calbiochem, San Diego, CA).
CO exposures. Cells were exposed to compressed air or 250 parts/million (ppm) CO. CO at a concentration of 1% (10,000 ppm) in compressed air was mixed with compressed air with 5% CO2 in a stainless steel mixing cylinder before delivery into the exposure chamber. Flow into the 1.2-ft2 cell culture chamber was maintained at a flow of 2 l/min. The cell culture chamber was humidified and maintained at 37°C. A CO analyzer (Interscan, Chatsworth, CA) was used to measure CO levels continuously in the chambers. Gas samples were taken by the analyzer through a port in the top of the chamber at a rate of 1 l/min and analyzed with electrochemical detection, with a sensitivity of 10-600 ppm. Concentration levels were measured hourly, and there were no fluctuations in the CO concentration once the chamber had equilibrated (~5 min).
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RESULTS |
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Tet-regulated expression of HO-1 in L929 fibroblast cells. E8.2.A3 cells, a glucocorticoid receptor-negative subclone of L929 cells (14), were stably transfected with 10 µg of plasmid pUHD15-1neo, and six G-418-resistant (600 µg/ml) clones (E8.T1 to E8.T6) were selected for further analysis. Duplicate cultures of each of these cells were transiently transfected with 2 µg of pUHDluc, incubated for 48 h in the presence and absence of Tet (1 µg/ml) and collected for measurement of luciferase activity (10). E8.T4 cells exhibited the highest level of Tet responsiveness (~75-fold induction of luciferase activity in the absence of Tet). These cells were stably transfected with 10 µg of the plasmid pToG/HO-1 or pToG/OH-1 with 1 µg of pPUR, and eight double-resistant (600 µg/ml of G-418 plus 5 µg/ml of puromycin) clones were collected and expanded. All antisense and six of eight sense (L929 HO-1) clones exhibited Tet-regulated expression of rat HO-1 sequences; those demonstrating the highest level of induction (as judged by RNA blot analysis) were used in subsequent studies.
L929 HO-1 cells were cultured for 24 h in the absence and presence of
varying concentrations of Tet, and HO-1 expression was monitored by
Northern blot analysis (Fig. 1A).
No HO-1 mRNA was detected in cells grown in the presence of Tet at 1 µg/ml, but it starts to be detected at 0.01 µg/ml; maximal HO-1
mRNA accumulation was observed in the absence of Tet. HO-1 protein is
also markedly increased when cells are exposed to medium lacking Tet
(Fig. 1B).
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TNF- induces apoptosis in L929 cells.
TNF-
-mediated apoptosis of L929 cells is extensively documented (6,
43). Initially, we confirmed that the L929 clone used in this study is
similarly sensitive to TNF-
. L929 HO-1 Tet+ cells were exposed to
varying concentrations of TNF-
, and apoptosis was assessed by flow
cytometry with the TUNEL assay. Treatment of L929 HO-1 Tet+ cells with
TNF-
resulted in apoptosis in a dose-dependent manner (Fig.
2). An increase in apoptotic cells (percent
gated cells) was observed at TNF-
concentrations of 1 (3.02%; Fig.
2B), 10 (12.69%; Fig. 2C), 50 (40.99%; Fig.
2D), and 100 (41.33%; Fig. 2E) ng/ml compared with
that in untreated control cells (0.58%; Fig. 2A). We further confirmed TNF-
-induced apoptosis in L929 fibroblasts by DNA
laddering (Fig. 3A) and nucleosomal assays
(Fig. 3B).
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HO-1 overexpression inhibits TNF--induced apoptosis
in L929 cells. To examine the effect of HO-1 induction on
apoptosis, L929 HO-1 Tet+ and L929 HO-1 Tet
cells were exposed
to TNF-
(100 ng/ml) for 24 h and then assessed for apoptosis by flow
cytometry or TUNEL assay. HO-1 overexpression had a marked
antiapoptotic effect: in L929 HO-1 Tet+ cells, apoptosis was readily
evident after TNF-
treatment (43.4% apoptotic cells) compared with
that in untreated L929 HO-1 Tet+ cells (2.6% apoptotic cells; Fig. 4). In contrast, the L929 HO-1 Tet
cells exhibited resistance to apoptosis even when exposed to a high
concentration of TNF-
(100 ng/ml; Fig. 4). This inhibitory effect of
HO-1 overexpression on apoptosis was also confirmed by electron
microscopy studies: L929 HO-1 Tet+ cells exhibited changes in cellular
morphology consistent with apoptosis, including chromatin margination,
presence of clefts in the cellular cytoplasm, and formation of
apoptotic bodies after TNF-
treatment (data not shown). The
occurrence of these apoptotic features was not observed in the L929
HO-1 Tet
cells after exposure to TNF-
. To confirm the
inhibitory role of HO-1 on TNF-
-induced apoptosis, we used L929
cells transfected with a Tet regulatory system linked to antisense HO-1
(L929 asHO-1 Tet+ and Tet
cells); marked apoptosis was
observed in these cells when they were exposed to TNF-
in the
presence (41.0% apoptotic cells) and absence (42.1% apoptotic cells)
of Tet in the culture medium as illustrated in Fig.
5. Furthermore, we treated the L929 HO-1
Tet
cells with TNF-
in the presence of SnPP, a selective inhibitor of HO activity. L929 HO-1 Tet
cells when exposed to SnPP (10 µM) exhibited a fourfold increase in apoptotic cells (16%)
compared with that in L929 HO-1 Tet
cells alone (4%), after TNF-
treatment.
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Possible mechanism by which HO-1 inhibits apoptosis. The
mechanism by which HO-1 overexpression confers protection against apoptosis is not clear. We hypothesized that CO, a major catalytic by-product of the HO-1 catalysis of heme, mediates this antiapoptotic effect. L929 HO-1 Tet cells were treated with TNF-
in the
presence and absence of a low concentration of CO exposure. As
expected, cells treated with TNF-
alone exhibited a marked increase
in apoptotic cells as assessed by nucleosomal assay (Fig.
6). In marked contrast, cells exposed to
TNF-
in the presence of CO did not demonstrate increased levels of
apoptosis (Fig. 6). We then examined whether inhibiting guanylyl
cyclase, a major target enzyme for CO, would ablate the antiapoptotic
effect of CO. Treatment of L929 HO-1 Tet
cells with ODQ (10 µM), an inhibitor of guanylyl cyclase, accentuated TNF-
-induced
apoptosis (250% increase; Fig. 7) compared
with that in L929 HO-1 Tet
cells alone.
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DISCUSSION |
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Many agents or environmental conditions that elicit cellular production
of ROS are potent inducers of the HO-1 gene; stimulation of
HO-1 provides protection against cellular oxidant stress both in vivo
and in vitro (9, 21, 27, 28, 40). The cellular and physiological bases
by which HO-1 exerts protection against oxidative stress are not clear
at this time. ROS have also been implicated in the apoptosis process
(32, 33, 42). Considering these observations and the fact that
antioxidants are known to inhibit the apoptotic pathway (22, 39), we
hypothesized that one possible pathway by which HO-1 confers protection
against oxidant injury is via its ability to impart antiapoptotic
activity. In this report, we show that overexpression of HO-1 inhibits
oxidant-mediated TNF--induced apoptosis of L929 fibroblast cells and
that exposure to a low concentration of CO likewise inhibits
TNF-
-induced apoptosis. Taken together, our data implicate CO as a
possible mediator of HO-1-induced protection against TNF-
-induced apoptosis.
ROS are involved in apoptotic cellular death; they can both trigger and
modulate the apoptotic process. Not surprisingly, antioxidants can
prevent apoptosis (20, 22, 39) in a cell-type and stimulus-specific
manner (16, 26, 34). Some proposed mechanisms by which antioxidants
inhibit apoptosis include free radical scavenging, metal chelation, and
modification of the thiol groups on proteins. TNF--induced apoptosis
of L929 cells has been extensively studied, ROS generation has been
implicated in the apoptotic process, and antioxidants protect against
TNF-
-induced cell death (32, 37, 42, 43). Thus in this model, the
antiapoptotic effect of HO-1 overexpression might be due to its end
products, namely the generation of bilirubin and ferritin. Bilirubin
and ferritin possess potent antioxidant properties both in vitro and in
vivo (4, 9, 41). Interestingly though, our observations that a low
concentration of CO protects cells against programmed cell death opens
a new avenue of investigation(s) into the mechanism(s) by which HO-1
provides protection against oxidative stress.
It is intriguing that CO at a low concentration provided a similar antiapoptotic effect as observed in HO-1-overexpressing cells. However, mammalian cells have the ability to generate endogenous CO primarily through the catalysis of heme by the enzyme HO (9, 15). The total cellular production of CO is generated primarily via heme degradation by HO (9). Furthermore, against the known paradigm of CO toxicity at high concentrations, there has been renewed interest in recent years in CO with observations that low levels of CO can exert cellular and biological effects. For example, CO, akin to the gaseous molecule nitric oxide, plays important roles in mediating neuronal transmission and regulating vasomotor tone (9, 15, 25). Otterbein et al. (29) have recently reported that a low concentration of CO (at the same dose used in this study) can provide protection against hyperoxic lung injury in vivo. Although the mechanism by which CO provided protection against lethal hyperoxia in vivo was not clear, it is interesting to note that animals exposed to a low concentration of CO exhibited significant attenuation of hyperoxia-induced lung apoptosis (28). We have also observed similar attenuation of oxidant-induced lung apoptosis in vivo in animals receiving exogenous HO-1 by gene transfer (29). Thus the in vitro antiapoptotic effects we observed in this study correlate with the in vivo antiapoptotic effect of CO and HO-1. These antiapoptotic effects of CO are intriguing, with reported observations that the gaseous molecule nitric oxide can also exert potent antiapoptotic effects (8, 17).
The mechanism by which HO or CO can exert an antiapoptotic function at
this time is not clear. It is believed that CO exerts biological
effects via a guanylyl cyclase or cGMP pathway in the vascular and
neuronal systems (15, 25). This pathway may also be involved in the
antiapoptotic effects of HO-1 and CO based on our observations that
ODQ, a guanylyl cyclase inhibitor, ablated the protective antiapoptotic
effects of HO-1 and that CO can increase cGMP levels in these
fibroblasts (data not shown). Alternative signaling pathway(s) may also
be involved in that important mediators such as nuclear factor-B and
the TNF death receptor pathway have been shown to be important in
mediating TNF-
-induced apoptosis (2, 3, 30, 36). Rigorous studies
are needed to investigate this plausible signaling pathway(s) by which
HO-1 or CO regulates its antiapoptotic effect.
In summary, this study unravels an important function of HO-1. The potent antiapoptotic effects observed in our report and recent accumulating data demonstrating the anti-inflammatory effects of HO-1 (28, 38) highlight the major mechanisms by which HO-1 serves to protect cells, organs, and host organisms against oxidative stress. Future studies are needed to address the signaling pathway by which this protection occurs.
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ACKNOWLEDGEMENTS |
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The work by A. M. K. Choi was supported by National Heart, Lung, and Blood Institute Grants HL-55330 and HL-60234; National Institute of Allergy and Infectious Diseases Grant AI-42365; and an American Heart Association Established Investigator Award. J. Alam was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-43135. I. Petrache and L. E. Otterbein were supported by a National Heart, Lung, and Blood Institute Multidisciplinary Training Grant.
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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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: A. M. K. Choi, Section of Pulmonary and Critical Care Medicine, Yale Univ. School of Medicine, 333 Cedar St., LCI 105, New Haven, CT 06520 (E-mail: augustine.choi{at}yale.edu).
Received 9 August 1999; accepted in final form 27 September 1999.
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