RAPID COMMUNICATION
Captopril inhibits apoptosis in human lung epithelial cells: a
potential antifibrotic mechanism
Bruce D.
Uhal1,
Claudia
Gidea1,
Raed
Bargout1,
Antonio
Bifero1,
Olivia
Ibarra-Sunga1,
Michael
Papp1,
Kevin
Flynn1, and
Gerasimos
Filippatos2
1 The Cardiovascular Institute,
Michael Reese Hospital and Medical Center, Chicago, Illinois 60612; and
2 Division of Cardiology,
Evangelismos General Hospital, GR-11526 Athens, Greece
 |
ABSTRACT |
The angiotensin-converting enzyme inhibitor
captopril has been shown to inhibit fibrogenesis in the lung, but the
mechanisms underlying this action are unclear. Apoptosis of lung
epithelial cells is believed to be involved in the pathogenesis of
pulmonary fibrosis. For these reasons, we studied the effect of
captopril on Fas-induced apoptosis in a human lung epithelial cell
line. Monoclonal antibodies that activate the Fas receptor induced
epithelial cell apoptosis as detected by chromatin condensation,
nuclear fragmentation, DNA fragmentation, and increased activities of caspase-1 and -3. Apoptosis was not induced by isotype-matched nonimmune mouse immunoglobulins or nonactivating anti-Fas monoclonal antibodies. When applied simultaneously with anti-Fas antibodies, 50 ng/ml of captopril completely abrogated apoptotic indexes based on
morphology, DNA fragmentation, and inducible caspase-1 activity and
significantly decreased the inducible activity of caspase-3. Inhibition
of apoptosis by captopril was concentration dependent, with an
IC50 of 70 pg/ml. These data
suggest that the inhibitory actions of captopril on pulmonary fibrosis
may be related to prevention of lung epithelial cell apoptosis.
pulmonary fibrosis; programmed cell death; type II pneumocyte; angiotensin-converting enzyme inhibitor
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INTRODUCTION |
EFFICIENT REPAIR OF DAMAGE to the alveolar epithelium
is believed to be essential for lung healing without fibrosis. An
investigation (27) of lung injury in animal models suggested that
incomplete or delayed alveolar repair leads to acceleration of collagen
deposition and lung fibroblast proliferation. Repair of the alveolar
epithelium is accomplished by the regulated proliferation and
differentiation of type II alveolar epithelial cells, which replace
both type II and type I alveolar epithelial cells lost to injury or
normal turnover (22). For these reasons, the type II alveolar
epithelial cell is believed to be a critical player in the pathogenesis
of pulmonary fibrosis (27, 19).
Apoptosis has been shown to be an important component of the
elimination of excess mesenchymal cells from diseased human lung (18)
and also is thought to be involved in the removal of excess epithelial
cells in remodeling hyperplastic human lung (2). On the other hand,
increased expression of Fas (APO1, CD95) and apoptosis was observed in
both bronchial and alveolar epithelial cells during the pathogenesis of
bleomycin-induced pulmonary fibrosis in mice (11). These observations
suggested that epithelial cell apoptosis might be involved in the
pathogenesis of lung fibrosis as well as in the resolution of
hypercellularity during healing of the lung. A critical role for lung
epithelial cell apoptosis as a pathogenic mechanism is supported by the
recent finding that ligation of the Fas receptor in vivo by
intratracheal administration of anti-Fas antibodies induced epithelial
cell apoptosis and pulmonary fibrosis in mice (10).
The angiotensin-converting enzyme (ACE) inhibitor captopril ameliorated
pulmonary fibrosis induced in rats by the plant alkaloid monocrotaline
(15). Captopril also inhibited the accumulation of collagens and mast
cells in irradiated rat lung (25). These observations suggested a novel
role for ACE inhibitors as modifiers of the response to pneumotoxicity,
but little is known about the cellular and molecular mechanisms
underlying these actions. Given the importance of epithelial integrity
in the pathogenesis of lung fibrosis, we hypothesized that captopril
might ameliorate lung fibrosis through direct inhibition of apoptosis
in lung epithelial cells. We report here that Fas-induced apoptosis of
human lung epithelial cells in culture is potently inhibited by
captopril at concentrations readily attained in vivo.
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METHODS |
Reagents and materials.
Monoclonal activating antibodies to human Fas (clone CH-11, purified
mouse IgM) were obtained from Upstate Biotechnology (Saranac Lake, NY).
Purified nonimmune mouse IgM was obtained from Sigma (St. Louis, MO).
Monoclonal nonactivating antibodies to human Fas (clones 5F-7 and 5F-9)
were obtained from Kamiya Biomedical (Seattle, WA). All other materials
were from sources described earlier (23) and were of reagent grade.
Cell culture. The human lung
adenocarcinoma cell line A549 was obtained from the American Type
Culture Collection and cultured in Ham's F-12 medium supplemented with
10% fetal bovine serum. Cells were seeded on 12-mm sterile glass
coverslips in 24-well chambers at a density of 20,000 cells/well. All
experiments were conducted at subconfluent densities of 80-90% in
Ham's F-12 medium supplemented with 1% fetal bovine serum. Antibodies
and captopril were diluted with Ham's F-12 medium and applied for 20 h
at 37°C in a 5% CO2
incubator.
Fluorescence detection of apoptosis.
Detection of apoptotic cells with propidium iodide was conducted as
described earlier (23) after digestion of ethanol-fixed cells with
DNase-free RNase in PBS containing 5 µg/ml of propidium iodide. In
these assays, detached cells were retained by centrifugation of the 24-well culture vessels during fixation with 70% ethanol. In situ end
labeling (ISEL) of fragmented DNA was conducted as described by
Gorczyca et al. (9). Briefly, ethanol was removed by rinsing coverslips
in distilled water for at least 10 min. The coverslips were then placed
in a saline-sodium citrate solution (0.3 M NaCl and 30 mM sodium
citrate in water, pH 7.0) at 37°C for 20 min. After four rinses in
PBS and four rinses in buffer A (50 mM
Tris · HCl, 5 mM
MgCl2, 10 mM
-mercaptoethanol,
and 0.005% BSA in water, pH 7.5), the coverslips were incubated at
18°C for 2 h with an ISEL solution (0.001 mM biotinylated dUTP, 20 U/ml of DNA polymerase I, and 0.01 mM each dATP, dCTP, and dGTP in
buffer A). Afterward, the sections
were rinsed thoroughly five times with buffer
A and three additional times with 0.5 M PBS.
Incorporated biotinylated dUTP was detected by incubation for 1 h at
37°C with avidin-rhodamine; the coverslips were then rinsed in
distilled water three times and mounted under Fluoromount solution
(Southern Biotechnology Associates, Birmingham, AL).
Caspase assays. The enzymatic
activities of caspase-1/interleukin-1
-converting enzyme (ICE)
and caspase 3/caspase protease protein-32 (CPP32)/Yama were
determined in intact A549 cell cultures preincubated under conditions
identical to those used for morphological assays of apoptosis. After 20 h of incubation with antibodies and/or captopril, the cells
were trypsinized from the culture vessels and resuspended in serum-free
culture medium containing anti-Fas antibodies and/or captopril
at the same concentrations as during the preincubation. After 1 h,
fluorogenic peptide substrates specific to each enzyme were added
separately to cuvettes containing the cell suspension for measurement
of total activity in intact cells. For measurement of caspase-1/ICE
activity, the peptide substrate
N-acetyl-Tyr-Val-Ala-Asp-7-amino-4-methylcoumarin
(Ac-YVAD-AMC, PharMingen, San Diego, CA) was used at 50 µM final
concentration. For caspase-3/CPP32/Yama, the peptide substrate
N-acetyl-Asp-Glu-Val-Asp-7-amino-4-methylcoumarin (Ac-DEVD-AMC, Upstate Biotechnology) was added at 200 µM final concentration. Production of fluorescent product over time was monitored in a spectrofluorometer at 380-nm excitation and 450-nm emission. Both enzyme assays were linear with time and protein concentration (data not shown).
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RESULTS |
Spontaneous or stimulated apoptosis was quantitated as described
earlier (23) in the human lung epithelial cell line A549 by
fluorescence detection of chromatin condensation and nuclear fragmentation (Fig. 1). By
this assay, apoptosis was induced in A549 cells by the monoclonal
activating antibody CH-11 (Fig.
2) but not by isotype-matched nonimmune
mouse IgM at an equivalent concentration. The monoclonal nonactivating
antibodies 5F-7 and 5F-9 also failed to induce apoptosis in A549 cells;
the latter results are consistent with recent findings by others (26)
using the same antibody preparations, which did not activate Fas in the
absence of other inducers.

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Fig. 1.
Fluorescence assay of apoptosis in human lung epithelial cells. Human
lung epithelial cell line A549 was cultured on glass coverslips as
described in METHODS. At end of test
period, detached cells were retained by centrifuging culture vessel
during fixation of cells in 70% ethanol. Fixed cells were incubated
for 30 min with 5 µg/ml of propidium iodide in PBS containing
DNase-free RNase; under these conditions, red fluorescence (>570 nm)
is specific for DNA. Field displayed is from Fas-activated cells (see
Fig. 2). As in an earlier work by Uhal et al. (23), apoptotic cells
were identified by presence of discrete nuclear fragments containing
condensed chromatin (arrow).
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Fig. 2.
Induction of apoptosis by monoclonal antibodies to Fas. Human lung A549
cells were cultured for 20 h in presence of equivalent concentrations
of monoclonal antibody CH-11 (Fas MAb), purified IgM fraction of
nonimmune mouse immunoglobulin (IgM), monoclonal antibody 5F-7 (MAb2),
and monoclonal antibody 5F-9 (MAb3). CONT, control (no additives).
Apoptotic cells were scored by nuclear morphology assay discussed in
Fig. 1. Values are means ± SE of at least 4 determinations.
* P < 0.01 compared with CONT
by ANOVA and Dunnett's test.
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The more sensitive assay of ISEL of fragmented DNA (Figs.
3 and 4)
revealed a potent inhibitory action of captopril on Fas-induced cell
death in A549 cells. Although captopril alone had no significant effect
on basal ISEL labeling, 50 ng/ml of captopril essentially abolished the
generation of ISEL-positive cells in response to ligation of Fas
antigen (Fig. 4). The same concentration of captopril also abrogated
Fas-induced stimulation of caspase-1/ICE activity (Fig.
5A) and
significantly inhibited Fas-induced activity of caspase-3/CPP32/Yama
(Fig. 5B), both cysteine proteases
believed to be critical in the execution of apoptosis (16). The effect of captopril on apoptosis was concentration dependent (Fig.
6); inhibition of Fas-induced ISEL labeling
was measurable at a concentration of 5 pg/ml of captopril and exhibited
an IC50 of 70 pg/ml. Inhibition of
apoptosis was maximal at 50 ng/ml of captopril, a concentration known
to be physiologically attainable in humans and maximally inhibitory for
ACE (7).

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Fig. 3.
In situ end labeling (ISEL) assay for DNA fragmentation. Ethanol-fixed
A549 cells on glass coverslips were subjected to ISEL labeling of
fragmented DNA as described in
METHODS. DNA fragments labeled with
biotinylated dUTP were detected with rhodamine-conjugated avidin.
A, C,
and E, phase-contrast images of the
same field of cells photographed under fluorescence illumination
(B,
D, and
F, respectively).
A and
B: CONT incubation without added
antibody. C and
D: 500 ng/ml of Fas MAb was applied
under same conditions as in Fig. 1. E
and F: Fas MAb was applied in presence
of 50 ng/ml of captopril. See Fig. 4 for quantitation of ISEL-positive
cells.
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Fig. 4.
Inhibition of Fas-induced DNA fragmentation by captopril. ISEL-positive
A549 cells were quantitated in at least 4 separate culture vessels
exposed with (+) and without ( ) indicated test materials as
described in Fig. 3. Concentrations were 500 ng/ml for Fas MAb and 50 ng/ml for captopril. Values are means ± SE of at least 4 determinations. * P < 0.001 compared with CONT by ANOVA and Dunnett's test.
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Fig. 5.
Captopril inhibits Fas-induced stimulation of caspase activities in
human lung epithelial cells. A549 cells were exposed as described in
Fig. 2 to Fas MAb alone or in presence of 50 ng/ml of captopril. Cells
were then analyzed in suspension culture for activity of
caspase-1/interleukin-1 -converting enzyme ICE with peptide substrate
Ac-YVAD-AMC (A) or caspase-3/caspase
protease protein-32/Yama with peptide substrate Ac-DEVD-AMC
(B) as described in
METHODS. Values are means ± SD of
at least 4 separate determinations. Significant difference
(P < 0.01) compared with:
* CONT; ** Fas MAb (by ANOVA and Student-Newman-Keuls
test).
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Fig. 6.
Concentration dependence of captopril inhibition of Fas-induced
apoptosis. A549 cells were exposed to Fas MAb as described in Fig. 2 in
presence of indicated concentrations of captopril. Inhibition of
apoptosis was detected as decrease in ISEL-positive cells, scored as
described in Fig. 4. IC50 = 0.07 ng/ml. Values are means ± SE of at least 6 determinations.
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DISCUSSION |
Captopril has been shown to ameliorate radiation-induced fibrosis of
the lung (25) and kidney (5), as well as lung fibrosis induced by the
plant alkaloid monocrotaline (15). Examinations of human lung
fibroblasts in culture suggest that this action might be due to direct
inhibitory effects on fibroblast proliferation, which were observed in
the presence of the mesenchymal cell mitogen basic fibroblast growth
factor (17). Captopril is also known to inhibit the proliferation of
cultured human mammary duct carcinoma cells (21) and to slow the growth
rate of experimental fibrosarcomas in rats (24). Direct inhibition of
zinc-dependent 72- and 92-kDa metalloproteinases produced by
endothelial cells has also been reported (24). Inhibition of
mesenchymal cell proliferation and metalloproteinase activities are
both potential antifibrotic mechanisms.
Our data suggest a complementary but entirely different mechanism of
antifibrotic potential by captopril. Numerous works have suggested a
relationship between incomplete epithelial repair and fibrogenesis
within the underlying interstitium in both experimental animal models
of lung injury (1, 12, 27) and biopsy specimens from patients with
fibrotic lung disease (13). In addition to providing intact barrier
functions, the alveolar epithelium (4) is believed to be an important
constitutive producer of prostaglandin E2, an inhibitor of fibroblast
proliferation (3). Normal alveolar epithelial cells also produce
urokinase-type plasminogen activator and plasminogen activator
inhibitor-1 and exhibit a potent capacity for the degradation of fibrin
clots in vitro (20). For these reasons, the loss of normal alveolar
epithelium would be expected to decrease the capacity of the alveolus
to clear intra-alveolar fibrin and would also deepen the commitment to
fibrogenesis by eliminating a known source of inhibitors of lung
fibroblast proliferation.
Apoptosis is now known to be an important regulator of alveolar
epithelial cell number in vitro (22, 23) and is a prominent feature of
the fibrotic lung in vivo (2, 11, 14). Expression of functional Fas has
been demonstrated in alveolar epithelial cells of the mouse and rat
lung in vitro (8) and in vivo (11). Furthermore, lung infiltration by
lymphocytes expressing Fas ligand has been speculated to contribute to
the induction of alveolar epithelial cell apoptosis observed after
intratracheal instillation of bleomycin in mice (11). The demonstration
that intratracheal administration of activating antibodies to Fas was
capable of inducing pulmonary fibrosis in mice (10) supports the
contention that Fas-induced apoptosis of the epithelium is a critical
component of the pathogenesis of pulmonary fibrosis.
To our knowledge, this is the first demonstration of the inhibition of
apoptosis by an ACE inhibitor in a cell type not of hematopoietic
lineage. Deas et al. (6) recently demonstrated inhibition of
Fas-induced apoptosis in activated human peripheral T cells by
captopril and other thiol compounds but not by nonthiol antioxidants.
Those results led the authors to speculate that the inhibition of
T-cell apoptosis was the result of sulfhydryl redox regulation of
critical molecules involved in the apoptotic signaling cascade. The
caspases are cysteine proteases critical to the signaling of apoptosis
and sensitive to sulfhydryl redox poise (16). Our results show that at
least two of these enzyme activities, caspase-1/ICE and
caspase-3/CPP32/Yama, are inhibited in situ by exposure of the intact
cell to captopril. We are currently addressing the possibility that
captopril and other thiol compounds inhibit lung epithelial cell
apoptosis through direct inhibition of caspase-1 and -3 activities
and/or other cysteine proteases required for the induction of
apoptosis.
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ACKNOWLEDGEMENTS |
This work was supported by National Heart, Lung, and Blood
Institute Grant HL-45136 to B. D. Uhal and the Women's Board Endowment to the Research and Education Foundation of the Michael Reese Medical
Staff.
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FOOTNOTES |
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: B. D. Uhal, The Cardiovascular Institute,
Michael Reese Hospital, 2929 S. Ellis Ave., Rm. 405KND, Chicago, IL
60612.
Received 12 June 1998; accepted in final form 10 August 1998.
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