help button home button
AJRCMB
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Nakajoh, M.
Right arrow Articles by Sasaki, H.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Nakajoh, M.
Right arrow Articles by Sasaki, H.
American Journal of Respiratory Cell and Molecular Biology. Vol. 28, pp. 296-304, 2003
© 2003 American Thoracic Society
DOI: 10.1165/rcmb.4845

Retinoic Acid Inhibits Elastase-Induced Injury in Human Lung Epithelial Cell Lines

Mizue Nakajoh, Takeyasu Fukushima, Tomoko Suzuki, Mutsuo Yamaya, Katsutoshi Nakayama, Kiyohisa Sekizawa and Hidetada Sasaki

Department of Geriatric and Respiratory Medicine, Tohoku University School of Medicine, Sendai; and Department of Respiratory Medicine, Institute of Clinical Medicine, University of Tsukuba, Tsukuba, Japan

Address correspondence to: Hidetada Sasaki, M.D., Professor and Chairman, Department of Geriatric and Respiratory Medicine, Tohoku University School of Medicine, 1-1 Seiryo-machi, Aoba-ku, Sendai, 980-8574 Japan. E-mail: dept{at}geriat.med.tohoku.ac.jp


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The protective effects of retinoic acid on elastase-induced lung epithelial cell injury were studied using elastase extracted from purulent human sputum, the BEAS-2B human bronchial epithelial cell line, A549 human type II lung cell line, and primary cultures of human tracheal epithelial cells. Elastase decreased viability of BEAS-2B cells, A549 cells, and human tracheal epithelial cells in concentration- and time-dependent fashions. Elastase also induced apoptosis of BEAS-2B cells, A549 cells, and the tracheal epithelial cells detected with cell death detection enzyme-linked immunosorbent assay and terminal deoxyribonucleotidyl transferase-mediated dUTP-biotin nick-end labeling (TUNEL) methods. Retinoic acid alone did not affect the viability of BEAS-2B cells, A549 cells, or the tracheal epithelial cells, and did not induce apoptosis of the cells. However, retinoic acid prevented the decreases in the viability and reduced apoptosis of BEAS-2B cells, A549 cells, and the tracheal epithelial cells induced by elastase. Likewise, retinoic acid inhibited caspase 3 activity in BEAS-2B cells and A549 cells induced by elastase, as well as proteolytic activity of elastase. Furthermore, caspase 3 inhibitor inhibited the elastase-induced apoptosis of the cells. These findings suggest that retinoic acid may inhibit elastase-induced lung epithelial cell injury partly through the inhibition of proteolytic activity of elastase and through the inhibition of caspase 3 activity by elastase. Retinoic acid may, therefore, have protective effects against the elastase-induced lung injury and subsequent development of pulmonary emphysema.

Abbreviations: Dulbecco's modified Eagle's medium, DMEM • dimethyl sulfoxide, DMSO • enzyme-linked immunosorbent assay, ELISA • fetal calf serum, FCS • human tracheal epithelial cells, HTE cells • phosphate-buffered saline, PBS • terminal deoxyribonucleotidyl transferase-mediated dUTP-biotin nick-end labeling, TUNEL


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Chronic obstructive pulmonary disease, which includes chronic pulmonary emphysema, chronic airway obstruction, and chronic bronchitis (1), is one of the leading causes of death worldwide, with an increasing prevalence and mortality (2). Cigarette smoke is the most common identifiable risk factor for chronic obstructive pulmonary disease, and two current hypotheses, the endogenous protease/antiprotease theory (3–6) and the oxidant/antioxidant theory (7, 8), have been established in the pathogenesis of chronic pulmonary emphysema.

To understand the pathogenesis of pulmonary emphysema, animal models of emphysema using elastase have been established (9, 10). Intratracheal instillation of elastase induces perivascular edema and an alveolar hemorrhage in the lung at the initial stage. The elastic framework of the lung is subsequently disrupted and the alveoli are enlarged and distorted (9, 10). Because elastase damages the airway epithelial cells and vascular endothelial cells (11–13), the hemorrhagic lung injury after intratracheal elastase instillation (9, 10) may be, in part, associated with the subsequent development of pulmonary emphysema, although damage to connective-tissue-matrix components of the lung induced by elastase are major causes of pulmonary emphysema (3–5). Furthermore, a recent report suggested the relation between lung cell apoptosis and emphysema in rats (14).

Massaro and coworkers (15) recently reported that retinoic acid inhibits elastase-induced pulmonary emphysema in rats. Retinoic acid has a variety of biological activities in the growth and differentiation of epithelial cells and in the development of pulmonary alveoli (16–19). Retinol storage in the fibroblast in the alveolar wall is related to the formation of alveolar septa (19). Although retinoic acid induces apoptosis of various cells (20, 21), it inhibits hydrogen peroxide–induced apoptosis in mesangeal cells (22). However, the mechanisms of the inhibitory effects of retinoic acid on elastase-induced pulmonary injury and subsequent development of pulmonary emphysema in rats have not been studied.

In the present study, we examined whether retinoic acid inhibits the injury and apoptosis of the cells induced by elastase, and studied the mechanisms responsible for the inhibitory effects of retinoic acid using the BEAS-2B human bronchial epithelial cell line, the A549 human type II epithelial cell line, and the primary cultures of human tracheal epithelial (HTE) cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Media Components
Reagents for cell culture media were obtained as follows: Dulbecco's modified Eagle's medium (DMEM), Ham's F-12 medium, phosphate-buffered saline (PBS), and fetal calf serum (FCS) were from GIBCO-BRL Life Technologies (Palo Alto, CA); trypsin, EDTA, penicillin, streptomycin, gentamicin, cholera toxin, and all trans retinoic acid were from Sigma (St. Louis, MO); insulin, transferrin, epidermal growth factor, endothelial cell growth supplement, hydrocortisone, and triiodothyronine were from Becton Dickinson (Collaborative Research Brand; Franklin Lakes, NJ); and elastase from purulent human sputum was from Elastin Products (Owensville, MO). Elastase solution used in the present study contained 875 U/mg protein. Elastase was purified from human sputum by ion exchange and affinity chromatography methods (personal communications from Elastin Products). Purity of the elastase was greater than 95% by SDS-PAGE, and the elastase did not contain elastase inhibitor or significant levels of endotoxin (< 10 EU) measured with the E-Toxate Multiple Test (personal communications from Elastin Products).

Human Epithelial Cell Culture
The BEAS-2B cells were supplied by Dr. Cutis Harris of the National Institutes of Health (Bethesda, MD). The A549 cells were supplied by the Cell Resource Center for Biomedical Research, Institute of Development, Aging and Cancer, Tohoku University.

The BEAS-2B cells (1 x 106 cells) were cultured in T25 flasks (Costar Corning, Cambridge, MA) in a serum-free medium consisting of DMEM–Ham's F-12 medium (50/50, vol/vol) and the following growth factors: 10 µg/ml of insulin, 5 µg/ml of transferrin, 25 ng/ml of epidermal growth factor, 7.5 µg/ml of endothelial cell growth supplement, 20 ng/ml of triiodothyronine, 0.36 µg/ml of hydrocortisone, and 20 ng/ml of cholera toxin. The A549 cells were cultured in T25 flasks in DMEM supplemented with 8% FCS. The cell culture medium was supplemented with 105 U/liter of penicillin, 100 mg/liter of streptomycin, and 50 mg/liter of gentamicin. After cells made confluent cell sheets, cells were collected by trypsinization (0.05% trypsin and 0.02% EDTA), replaced in cell culture medium and antibiotics in 96-well plates (4 x 104/0.2 ml) and cultured at 37°C in 5% CO2–95% air. When A549 cells made confluent sheets in 96-well dishes, the cells were rinsed with PBS and further cultured in 200 µl of the DMEM–Ham's F-12 with growth factors.

The HTE cells were isolated and plated in 96-well dishes with the methods as previously described (23), in 200 µl of the DMEM–Ham's F-12 with 2% Ultroser G serum substitute (USG; BioSepra, Marlborough, MA).

Assessment of Cell Viability
Cell viability was assessed with colorimetric MTT (tetrazolium) assay with a Cell Counting Kit (Dojindo, Kumamoto, Japan) (24). BEAS-2B cells and A549 cells cultured in 200 µl of the DMEM–Ham's F-12 with growth factors, and primary cultures of HTE cells cultured in 200 µl of the DMEM–Ham's F-12 with 2% USG in 96-well dishes were treated with elastase in the presence or absence of retinoic acid for 24 h. Cells were then treated with 10 µl of tetrazolium solution (pH 7.4) containing 16.3 mg/5 ml of 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium (WST-1), 20 mM HEPES, and 0.2 mM 1-methoxyl-5-methylphenazinium methylsulfate (1-methoxyl PMS). The cells in the dishes were then incubated at 37°C for 1 h in 5% CO2–95% air. The absorbance intensity of the solution was measured on a spectrophotometer (Labsystems Multiskan BICHROMATIC; Labsystems, Helsinki, Finland) using a test wavelength of 450–690 nm.

Assessment of DNA Fragmentation by Cell Death Detection Enzyme-Linked Immunosorbent Assay and TUNEL
Assessment of DNA fragmentation associated with apoptosis was performed by enzyme-linked immunosorbent assay (ELISA) with cell death detection ELISA (Boehringer Mannheim, Indianapolis, IN) as previous described (25). Either BEAS-2B cells, A549 cells, or HTE cells were treated with elastase in the presence or absence of retinoic acid for 24 h. The culture supernatants were collected by centrifugation (200 x g, 10 min) and stored at 4°C for the assay. To bind histone-associated DNA oligonucleosomes in supernatants to a biotinylated antihistone antibody, samples of supernatants were incubated in microtiter plates coated with streptavidin conjugated with biotinylated mouse antihistone antibody (clone H11–4). Plates were washed, and nonspecific binding sites were saturated with an anti-mouse DNA monoclonal antibody (MCA-33) and then conjugated with an anti-DNA antibody bound to peroxidase. To determine the amount of retained peroxidase, 2,2'-azino-di (3-ethylbenzthiazoline-6-sulfonate) (ABTS) was added as a substrate, and the complex was measured by spectrophotometer (Labsystems Multiskan BICHROMATIC; Labsystems) at 405 nm. Results are expressed as the ratio of sample absorbance to absorbance of the room air control sample measured daily.

Assessment of DNA fragmentation associated with apoptosis of BEAS-2B cells and A549 cells was also performed by TUNEL assay with the MEBSTAIN Apoptosis Kit (Medical and Biological Laboratories, Nagoya, Japan) as previously described (26). Cells were washed, fixed, permeabilized, and labeled with avidin-FITC and biotin-dUTP according to the manufacturer's instructions. The number of FITC-labeled cells was counted under a fluorescent microscopy (Meridian Instruments, Okemos, MI).

Assessment of Caspase 3 Activity
Assessment of caspase 3 activity was performed by fluorometric immunosorbent enzyme assay (FIENA) according to the instruction manual of the Caspase 3 Activity Assay Kit (Roche, Mannheim, Germany). BEAS-2B cells (2 x 106), cultured in 6-well culture dishes, were collected with scrapers and suspended in 1 ml of PBS in 15-ml centrifuge tubes (Corning). Pellets of the cells were collected by centrifugation (300 x g, 5 min, 4°C), resuspended in 1.5 ml of PBS and transferred to Eppendorf tubes. After centrifugation (600 x g, 5 min, 4°C), pellets of the cells were lysed in 200 µl of buffer containing 1x DTT, and incubated for 1 min at 4°C. The supernatants of cell lysates were collected by centrifugation at maximum speed in a table top centrifuge (13,000 rpm, 1 min) and were stored at 4°C for the assay. Samples of supernatants were incubated with microtiter plates absorbed with anti-caspase 3 (27). Plates were washed, and nonspecific binding sites were saturated with the blocking buffer of the Kit. Plates were conjugated with fluorescence substrate solution containing 50 µM of acetyl-Asp-Glu-Val-Asp-7-amino-4-trifluoromethyl-coumarin (Ac-DEVD-AFC), and incubated for 2 h at 37°C. Ac-DEVD-AFC is cleaved proportionally to the amount of activated caspase 3 and generates free fluorescent 7-amido-4-trifluoromethyl-coumarin (AFC). The content of free AFC was measured fluorometrically at 505 nm in excitation at 400 nm by a fluorescence reader (Fluoroskan; Labsystems). The fluorescence intensity is expressed as raw fluorescence intensity minus background fluorescence intensity.

Proteolytic Activity of Elastase
Specific proteolytic activity of elastase was determined by methoxysuccinyl -Ala-Ala –Pro-Val-p-nitroanilide (Sigma) hydrolysis by the method of Fujimoto and coworkers (28) with some modifications.

To study the effects of either retinoic acid or {alpha}1-antitrypsin on the proteolytic activity of elastase, 0.1 µl of retinoic acid, {alpha}1-antitrypsin, or the vehicle of inhibitors was added to the reaction mixture (100 µl) containing 75 µl of HEPES-NaCl buffer (0.1 M HEPES, 0.5 M NaCl, pH 7.5) and 25 µl of methoxysuccinyl-Ala-Ala-Pro-Val-p-nitroanilide (6 µg/ml). Methoxysuccinyl-Ala-Ala-Pro-Val-p-nitroanilide was dissolved in the HEPES-NaCl buffer containing 30% dimethyl sulfoxide (DMSO). Elastase was diluted to 600 U/ml in the HEPES-NaCl buffer. The reaction was started by adding 0.1 µl of elastase solution into the 100 µl of reaction mixture at the final concentration of 0.6 U/ml. The developed color was measured spectrophotometrically at 405 nm. One unit of elastase activity was defined as the quantity of enzyme that liberated 1 µmol of p-nitroanilide in 1 min.

Statistical Analysis
Results are expressed as mean ± SEM. Statistical analysis was performed using two-way repeated measures of ANOVA. Subsequent post hoc analysis was made using Bonferroni's method. For all analyses, values of P < 0.05 were assumed to be significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Cell Viability
The cell viability measured by colorimetric MTT assay was stable with time and more than 96% in BEAS-2B cells and 95% in A549 cells for 48 h. In the preliminary experiments, we found that elastase decreased the viability of BEAS-2B cells and A549 cells in a narrow range of concentrations between 0.01 U/ml and 1 U/ml in BEAS-2B cells and between 0.03 U/ml and 10 U/ml in A549 cells and HTE cells. Therefore, to examine the dose–response effects of elastase on the viability of BEAS-2B cells or A549 cells, the cells were treated with elastase at various concentrations ranging from 0.01–1 U/ml for either 6 or 24 h in BEAS-2B cells, and from 0.03–10 U/ml for either 6 or 24 h in A549 cells and HTE cells. The viability of A549 cells significantly decreased at a 1 U/ml concentration of elastase 6 h after administration, and at concentrations higher than 0.05 U/ml of elastase 24 h after administration (Figure 1A). Likewise, the viability of A549 cells significantly decreased at concentrations higher than 3 U/ml of elastase 6 h after and at concentrations higher than 0.1 U/ml of elastase 24 h after administration (Figure 1B). The viability of the primary cultures of HTE cells significantly decreased at concentrations of 10 U/ml of elastase 6 h after administration, and at concentrations higher than 1 U/ml of elastase 24 h after administration (Figure 1C). The effects of elastase on cell viability were dependent on concentration in BEAS-2B cells, A549 cells, and human tracheal epithelial cells (P < 0.05 in each by ANOVA).



View larger version (25K):
[in this window]
[in a new window]
  		Figure 1. Concentration-response effects of elastase on viability of BEAS-2B cells (A), A549 cells (B), and primary cultures of HTE cells (C) 6 h (open columns) or 24 h (filled columns) after treatment with elastase. Viability was normalized to the absorbance intensity of tetrazolium in the BEAS-2B cells, A549 cells, or primary cultures of HTE cells in medium alone (control) and expressed as % of control. Results are mean ± SEM from five samples. Significant differences from control values are indicated by *P < 0.05, **P < 0.01, and ***P < 0.001.

 
Furthermore, we found in the preliminary experiments that treatment with 0.06, 0.3, and 3 U/ml of elastase for 24 h consistently reduced cell viability in BEAS-2B cells, A549 cells, and HTE cells, respectively. Therefore, to examine the dose–response effects of retinoic acid on decreases in the viabilty of BEAS-2B cells, A549 cells, or primary cultures of HTE cells by elastase, the cells were treated with elastase (0.06 U/ml in BEAS-2B cells, 0.3 U/ml in A549 cells, and 3 U/ml in HTE cells) for 24 h in the presence or absence of retinoic acid at various concentrations ranging from 10-10 to 10-6 M.

Treatment with retinoic acid itself (from 10-10 to 10-6 M) did not alter the viability of BEAS-2B cells (Figure 2A), A549 cells (Figure 2B), or primary cultures of HTE cells (Figure 2C) for 24 h. However, retinoic acid inhibited decreases in cell viability induced by elastase in BEAS-2B cells (0.06 U/ml of elastase) (Figure 2D), A549 cells (0.3 U/ml of elastase) (Figure 2E), and HTE cells (3 U/ml of elastase) (Figure 2F).



View larger version (19K):
[in this window]
[in a new window]
  		Figure 2. Concentration-response effects of retinoic acid (24 h) on the viability of BEAS-2B cells (A), A549 cells (B), and primary cultures of HTE cells (C). Concentration-response effects of retinoic acid on the viability of BEAS-2B cells (D), A549 cells (E), and primary cultures of HTE cells (F) 24 h after treatment with elastase (0.06 U/ml in BEAS-2B cells, 0.3 U/ml in A549 cells, and 3 U/ml in HTE cells). Viability was normalized to the absorbance intensity of tetrazolium in the cells in medium alone (control), and expressed as % of control. Results are mean ± SEM from five samples. Significant differences from elastase alone are indicated by *P < 0.05 and **P < 0.01.

 
Furthermore, to examine that the inhibitory effects of retinoic acid on elastase-induced cell injury are not caused by in vitro biochemical effects, elastase (0.06 U/ml in BEAS-2B cells, 0.3 U/ml in A549 cells) and either retinoic acid (10-8 M) or vehicle of retinoic acid (0.001% DMSO in HEPES-NaCl buffer) were preincubated at 37°C for 24 h, and then added to the cells. When elastase and retinoic acid were preincubated, retinoic acid also significantly inhibited the decreases in viability of the BEAS-2B cells and A549 cells induced by elastase. The viability after treatment with elastase plus vehicle of retinoic acid was 62 ± 4% in BEAS-2B cells (n = 3) and 55 ± 3% in A549 cells (n = 3), and that after treatment with elastase plus retinoic acid was 92 ± 6% in BEAS-2B cells (n = 3, P < 0.05) and 73 ± 4% in A549 cells (n = 3, P < 0.05).

Likewise, when retinoic acid (10-8 M) was preincubated with the cells at 37°C for 24 h, and then the elastase (0.06 U/ml in BEAS-2B cells, 0.3 U/ml in A549 cells) was added to the cells after washing out the retinoic acid, pretreatment with retinoic acid significantly inhibited the decreases in viability of the BEAS-2B cells and A549 cells induced by elastase. The viability after treatment with elastase was 48 ± 3% in BEAS-2B cells (n = 3) and 51 ± 4% in A549 cells (n = 3), and that after treatment with elastase in the presence of retinoic acid pretreatment was 82 ± 5% in BEAS-2B cells (n = 3, P < 0.05) and 63 ± 3% in A549 cells (n = 3, P < 0.05).

To examine the effects of culture supernatants released from the cells after treatment with retinoic acid, retinoic acid (10-8 M) was preincubated with the cells at 37°C, and culture supernatants were collected 24 h after the cells were cultured in the medium alone. When the cells were exposed to the elastase (0.06 U/ml in BEAS-2B cells, 0.3 U/ml in A549 cells) in the presence of culture supernatants after pretreatment of retinoic acid, the culture supernatants did not inhibit the decreases in viability of the BEAS-2B cells and A549 cells induced by elastase (data not shown).

DNA Fragmentation by Cell Death Detection ELISA and TUNEL
To examine the effects of retinoic acid on the elastase-induced DNA fragmentation in BEAS-2B cells, A549 cells, and HTE cells, cells were treated with elastase (0.06 U/ml in BEAS-2B cells, 0.3 U/ml in A549 cells, and 3 U/ml in HTE cells) for 24 h in the presence or absence of 10-8 M of retinoic acid. Levels of DNA fragmentation in BEAS-2B cells (Figure 3A), A549 cells (Figure 3B), and HTE cells (Figure 3C) assessed by cell death detection ELISA were low in either the medium alone or retinoic acid alone (10-8 M) for 24 h. Elastase (0.06 U/ml in BEAS-2B cells, 0.3 U/ml in A549 cells, and 3 U/ml in HTE cells) caused a significant increase in DNA fragmentation levels in BEAS-2B cells, A549 cells, and HTE cells. Retinoic acid (10-8 M, 24 h) significantly inhibited DNA fragmentation induced by elastase (0.06 U/ml in BEAS-2B cells, 0.3 U/ml in A549 cells, and 3 U/ml in HTE cells) in these three types of cells (Figure 3).



View larger version (14K):
[in this window]
[in a new window]
  		Figure 3. DNA fragmentation measured by cell death detection ELISA 24 h after treatment of BEAS-2B cells (A), A549 cells (B), or primary cultures of HTE cells (C) with elastase (EL, 0.06 U/ml in BEAS-2B cells, 0.3 U/ml in A549 cells, and 3 U/ml in HTE cells) or retinoic acid (RA, 10-8 M). Results are expressed as the ratio of sample absorbance to absorbance of room air control sample. Results are mean ± SEM from five samples. Significant differences from medium alone and elastase alone are indicated by *P < 0.05 and +P < 0.05, respectively.

 
Furthermore, to examine that the inhibitory effects of retinoic acid on elastase-induced cell injury are not caused by in vitro biochemical effects, elastase (0.06 U/ml in BEAS-2B cells, 0.3 U/ml in A549 cells) and either retinoic acid (10-8 M) or vehicle of retinoic acid (0.001% DMSO in HEPES-NaCl buffer) were preincubated at 37°C for 24 h, and then added to the cells. When elastase and retinoic acid were preincubated, retinoic acid also significantly inhibited the DNA fragmentation in viability of the BEAS-2B cells and A549 cells induced by elastase. The absorbance ratio assessed by cell death detection ELISA after treatment with elastase plus vehicle of retinoic acid was 1.82 ± 0.31 in BEAS-2B cells (n = 3) and 0.68 ± 0.08 in A549 cells (n = 3), and that after treatment with elastase plus retinoic acid was 1.42 ± 0.26 in BEAS-2B cells (n = 3, P < 0.05) and 0.51 ± 0.04 in A549 cells (n = 3, P < 0.05).

Likewise, when retinoic acid (10-8 M) was preincubated with the cells at 37°C for 24 h, and then the elastase (0.06 U/ml in BEAS-2B cells, 0.3 U/ml in A549 cells) was added to the cells after washing out the retinoic acid, pretreatment of retinoic acid significantly inhibited the DNA fragmentation of the BEAS-2B cells and A549 cells induced by elastase. The absorbance ratio assessed by cell death detection ELISA after treatment with elastase was 1.92 ± 0.32 in BEAS-2B cells (n = 3) and 0.77 ± 0.13 in A549 cells (n = 3), and that after treatment with elastase in the presence of retinoic acid pretreatment was 1.53 ± 0.21 in BEAS-2B cells (n = 3, P < 0.05) and 0.61 ± 0.03 in A549 cells (n = 3, P < 0.05).

To examine the effects of culture supernatants released from the cells after treatment with retinoic acid, retinoic acid (10-8 M) was preincubated with the cells at 37°C, and culture supernatants were collected 24 h after the cells were cultured in the medium alone. When the cells were exposed to the elastase (0.06 U/ml in BEAS-2B cells, 0.3 U/ml in A549 cells) in the presence of culture supernatants after pretreatment of retinoic acid, the culture supernatants did not inhibit the DNA fragmentation of the BEAS-2B cells and A549 cells induced by elastase (data not shown).

The inhibitory effects of retinoic acid on elastase-induced DNA fragmentation were also shown by TUNEL staining. Elastase (0.06 U/ml in BEAS-2B cells and 0.3 U/ml in A549 cells, 24 h) altered the color of fluorescence from red to green in BEAS-2B cells (Figures 4A and 4B) and A549 cells (Figures 4D and 4E), and caused increases in the number of cell deaths by apoptosis in BEAS-2B cells (Figure 4G) and A549 cells (Figure 4H). Retinoic acid (10-8 M, 24 h) inhibited elastase-induced increases in the number of cell deaths in BEAS-2B cells (Figures 4C and 4G) and A549 cells (Figures 4F and 4H).




View larger version (91K):
[in this window]
[in a new window]
  		Figure 4. (A–F) TUNEL staining of BEAS-2B cells (A, B, C) and A549 cells (D, E, F) in the presence (B, E) or absence (A, D) of treatment with elastase (0.06 U/ml in BEAS-2B cells and 0.3 U/ml in A549 cells, 24 h), or presence of both elastase and retinoic acid (10-8 M, 24 h) (C, F). Elastase increased, and retinoic acid decreased, the number of TUNEL-positive cells with green fluorescence. Bars indicate 20 µm. Data are representative of five different experiments. (G and H) The number of TUNEL-positive cells (%) in BEAS-2B cells (G) and A549 cells (H) in the presence or absence of treatment with elastase (EL, 0.06 U/ml in BEAS-2B cells and 0.3 U/ml in A549 cells, 24 h), or presence of both elastase and retinoic acid (RA, 10-8 M, 24 h). Results are mean ± SEM from five samples. Significant differences from medium alone and elastase alone are indicated by **P < 0.01 and ++P < 0.01, respectively.

 
Caspase 3 Activity
Because caspase 3 activity in BEAS-2B cells and A549 cells was stable for 5 d in medium alone after the cells made confluent cell sheets, the assay of caspase 3 activity was performed between Days 2 and 4. To examine the time course of caspase 3 activity in BEAS-2B cells after elastase exposure, cells were treated with elastase (0.06 U/ml in BEAS-2B cells, and 0.3 U/ml in A549 cells) for various periods from 2–10 h. The caspase 3 activity in BEAS-2B cells and in A549 cells increased with time after the addition of elastase (0.06 U/ml in BEAS-2B cells, and 0.3 U/ml in A549 cells), with the maximal increases at 6 h after treatment (Figure 5A). Therefore, to examine the effects of retinoic acid on the elastase-induced caspase 3 activation in BEAS-2B cells and A549 cells, cells were treated with elastase (0.06 U/ml in BEAS-2B cells and 0.3 U/ml in A549 cells) for 6 h in the presence or absence of retinoic acid at various concentrations ranging from 10-10 to 10-7 M after the cells made confluent cell sheets in 6-well culture dishes.





View larger version (53K):
[in this window]
[in a new window]
  		Figure 5. (A) Time course of the fluorecence intensity caused by caspase 3 activity in the BEAS-2B cells (open circles) and A549 cells (filled circles) after treatment with elastase (0.06 U/ml in BEAS-2B cells and 0.3 U/ml in A549 cells). (B and C) Effects of retinoic acid (RA, 10-8 M, 6 h) and elastase (EL, 0.06 U/ml in BEAS-2B cells and 0.3 U/ml in A549 cells, 6 h) on the fluorescence intensity caused by caspase 3 activity in the BEAS-2B (B) and A549 (C) cells, and concentration-response effects of retinoic acid (RA) on the fluorescence intensity caused by caspase 3 activity in the BEAS-2B (B) and A549 (C) cells after treatment with elastase (EL, 0.06 U/ml in BEAS-2B cells and 0.3 U/ml in A549 cells, 6 h). Cells were treated with elastase for 6 h in the presence or absence of retinoic acid. Results are mean ± SEM from five samples. Open bars, control; striped bars, retinoic acid; hatched bars, elastase; solid bars, elastase + retinoic acid. Significant differences from medium alone (control, C) are indicated by *P < 0.05, **P < 0.01. Significant differences from elastase alone (EL) are indicated by +P < 0.05 and ++P < 0.01.

 
Treatment of elastase alone (0.06 U/ml in BEAS-2B cells, and 0.3 U/ml in A549 cells, 6 h) increased caspase 3 activity in both BEAS-2B cells (Figure 5B) and A549 cells (Figure 5C). Retinoic acid inhibited elastase-induced increases in caspase 3 activity dose-dependently, as well as baseline caspase 3 activity in both BEAS-2B cells (Figure 5B) and A549 cells (Figure 5C).

Effects of Caspase 3 Inhibitor on the DNA Fragmentation
To examine the effects of caspase inhibitor on the elastase-induced DNA fragmentation of the cells, either BEAS-2B cells or A549 cells were treated with caspase 3 inhibitor (Ac-DEVD-CHO; BIOMOL, Plymouth Meeting, PA) (29) at various concentrations ranging from 10-6 to 10-4 M from 1 h before until 24 h after administration of elastase (0.06 U/ml in BEAS-2B cells, and 0.3 U/ml in A549 cells). DNA fragmentation levels in both BEAS-2B cells and A549 cells increased after the addition of elastase (0.06 U/ml in BEAS-2B cells, and 0.3 U/ml in A549 cells, 24 h) (Figures 6A and 6B). Caspase 3 inhibitor itself did not alter the DNA fragmentation in both cell lines. However, the caspase 3 inhibitor inhibited elastase-induced DNA fragmentation dose-dependently in both BEAS-2B cells (Figure 6A) and A549 cells (Figure 6B).



View larger version (22K):
[in this window]
[in a new window]
  		Figure 6. Effects of caspase 3 inhibitor (CI, 100 µM), elastase (EL, 0.06 U/ml in BEAS-2B cells and 0.3 U/ml in A549 cells), and concentration-response effects of caspase 3 inhibitor (CI) on the DNA fragmentation measured by cell death detection ELISA 24 h after treatment of BEAS-2B cells (A) or A549 cells (B) with elastase (EL, 0.06 U/ml in BEAS-2B cells and 0.3 U/ml in A549 cells). Cells were treated with caspase 3 inhibitor from 1 h before until 24 h after addition of elastase into the culture medium. Results are expressed as the ratio of sample absorbance to absorbance of room air control sample. Results are mean ± SEM from five samples. Open bars, control; striped bars, caspase inhibitor; hatched bars, elastase; solid bars, elastase + caspase inhibitor. Significant differences from medium alone (Control, C) and elastase alone (EL) are indicated by *P < 0.05 and +P < 0.05, respectively.

 
Proteolytic Activity of Elastase
Retinoic acid inhibited the elastase activity determined by methoxysuccinyl-Ala-Ala-Pro-Val-p-nitroanilide hydrolysis. To study the effects of retinoic acid on elastase activity (0.6 U/ml), retinoic acid at various concentrations ranging from 10-10 to 10-6 M, or the vehicle of retinoic acid, was added to the mixture. Retinoic acid was dissolved in DMSO and diluted in the HEPES-NaCl buffer. Likewise, to study the effects of {alpha}1-antitrypsin on elastase activity (0.6 U/ml), {alpha}1-protease inhibitor (Sigma) at various concentrations ranging from 10-9 to 10-5 M, or distilled water as the vehicle of {alpha}1-protease inhibitor, was added to the mixture.

Both retinoic acid (Figure 7A) and {alpha}1-protease inhibitor (Figure 7B) inhibited the elastase activity dose-dependently (P < 0.05, ANOVA).



View larger version (12K):
[in this window]
[in a new window]
  		Figure 7. Concentration-response effects of retinoic acid (A) and {alpha}-1 protease inhibitor ({alpha}-1-PI) (B) on elastase activity. Elastase activity was normalized to the absorbance intensity of p-nitroanilide in elastase alone (control), and expressed as % of control. Results are mean ± SEM from five samples. Significant differences from elastase alone (control) are indicated by *P < 0.05, **P < 0.01, and ***P < 0.001.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We showed that exposure of elastase decreases the viability of the BEAS-2B human bronchial epithelial cell line, the A549 human type II lung cell line, and primary cultures of HTE cells. The exposure of elastase also induced DNA fragmentation of these epithelial cells measured with cell death detection ELISA and TUNEL. Furthermore, treatment with retinoic acid significantly inhibited the decreases in the viability and the increases in DNA fragmentation induced by elastase exposure in BEAS-2B cells, A549 cells, and HTE cells. Preincubation of retinoic acid with the epithelial cells also inhibited elastase-induced decreases in the viability and apoptosis of the cells. When elastase was preincubated with retinoic acid, the inhibitory effects of retinoic acid were not affected, suggesting the inhibitory effects of retinoic acid on elastase might not be biochemical effects. Elastase used in the present study did not contain endotoxin or elastase inhibitor. Because {alpha}1-protease inhibitor reduced the proteolytic activity of elastase solution used in the present study, elastase-induced decreases in viability and increases in DNA fragmentation of the epithelial cells might be caused by elastase activity. Furthermore, culture supernatants in the epithelial cells pretreated with retinoic acid did not inhibit elastase-induced decreases in the viability and apoptosis of the cells, suggesting that the inhibitory effects of retinoic acid on the elastase-induced cell injury might not associated with the factors released from the cells treated with retinoic acid. However, it should be noted here that the concentrations of elastase used in the present study were significantly higher than those detected in bronchoalveolar lavage fluids in smokers (3).

Elastase, released from various cells in the lung, relates to the pathogenesis of human pulmonary emphysema (3, 4, 30). To understand the pathogenesis of pulmonary emphysema, animal models of emphysema using elastase have been established. Intratracheally instilled elastase produces a region closely resembling human pulmonary emphysema in hamster lungs (9, 10). Elastase induces perivascular edema and a hemorrhagic injury in the lung at the initial stage after intratracheal instillation (9, 10). Subsequently, the lungs become grossly emphysematous, the elastic framework of the lung is disrupted, and the alveoli are enlarged and distorted. Kuhn and coworkers (31) showed initial decreases in elastin content and subsequent increases in elastin and collagen synthesis after elastase instillation. These findings suggest that elastase-induced damage of connective-tissue-matrix components and subsequent synthesis of those components are major causes of pulmonary emphysema. On the other hand, elastase damages the airway epithelial cells and vascular endothelial cells (12, 13). Therefore, the hemorrhagic lung injury after intratracheal elastase instillation (9, 10) may be partly associated with the development of pulmonary emphysema.

In the present study, elastase reduced cell viability and induced DNA fragmentation in the human airway and alveolar epithelial cells. These findings are consistent with the previous reports demonstrating elastase-induced damage in airway epithelial cells and vascular endothelial cells (11–13, 21, 32). Although it is still uncertain whether apoptosis of lung cells is associated with the development of human pulmonary emphysema, marked apoptosis of type II pneumocytes was detected in the lung tissues from patients with acute lung injury (33). Furthermore, a recent report suggested the causal relation between lung cell apoptosis and emphysema in rats (14). The present study also showed that elastase increased the activity of caspase 3, which plays a key role during the apoptotic process (34) in BEAS-2B cells and in A549 cells, as demonstrated in neutrophils, leukemia cells (35, 36), and alveolar septal cells (14). Furthermore, caspase 3 inhibitor significantly inhibited the elastase-induced DNA fragmentation of both BEAS-2B and A549 cells. Therefore, the activation of caspase 3 might be partly associated with the apoptosis of epithelial cells in the present study.

There are conflicting reports on the effects of retinoic acid on apoptosis. Retinoic acid induces apoptosis and increases caspase activity in various cells (2, 30, 37), whereas it has no effect on T cell apoptosis induced by HIV-1 virus infection (38). Although little is known about the anti-apoptotic effect of retinoic acid, it inhibits hydrogen peroxide–induced apoptosis of mesangial cells (22). Thus, the effects of retinoic acid on the apoptosis may differ among cell types. In the present study, retinoic acid reduced the caspase 3 activity in BEAS-2B cells and A549 cells before and after exposure to elastase. Furthermore, we demonstrated that retinoic acid reduces proteolytic activity of elastase as shown by Sklan and coworkers (39), in which they suggested that a carboxyl group in retinoic acid is required to inhibit elastase activity (39). Retinoic acid also inhibits proteolysis of the von Willebrand factor and procoagulant, and fibrinolytic activities by elastase (13, 40). Therefore, retinoic acid might inhibit not only the elastase-induced caspase 3 activity, but also the elastase activity itself.

In summary, we have demonstrated that retinoic acid inhibited the elastase-induced decreases in the viability and the increases in DNA fragmentation of the BEAS-2B human airway epithelial cell line, the A549 human alveolar epithelial cell line, and primary cultures of HTE cells. Retinoic acid inhibited the elastase activity, and elastase-induced caspase 3 activity, in the cells. Pretreatment with retinoic acid also prevented the elastase-induced decreases in the viability, and reduced apoptosis of the cells. Furthermore, caspase 3 inhibitor inhibited the elastase-induced decreases in cell viability. These findings suggest that retinoic acid may inhibit elastase-induced lung cell damage, partly through the direct inhibition of proteolytic activity of elastase and through the inhibition of caspase 3 activity. Retinoic acid may therefore have protective effects against the development of pulmonary emphysema.


   Acknowledgments
 
The authors thank Mr. Grant Crittenden for the English correction.

Received in original form February 19, 2002

Received in final form September 11, 2002


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Celli, B. R., G. L. Snider, J. Heffner, B. Tiep, I. Ziment, B. Make, S. Braman, G. Olsen, and Y. Phillips. 1995. Standards for the diagnosis and care of patients with chronic obstructive pulmonary disease. Am. J. Respir. Crit. Care Med. 152:S77–S120.
  2. Thom, T. J. 1989. International comparisons in COPD mortality. Am. Rev. Respir. Dis. 140:S27–S34.[Medline]
  3. Janoff, A. 1985. Elastases and emphysema: current assessment of the protease-antiprotease hypothesis. Am. Rev. Respir. Dis. 132:417–433.[Medline]
  4. Snider, G. L. 1992. Emphysema: the first two centuries-and beyond: a historical overview, with suggestions for future research. Part 2. Am. Rev. Respir. Dis. 146:1615–1622.[Medline]
  5. Hoidal, J. R., and D. E. Niewoehner. 1983. Pathogenesis of emphysema. Chest 83:679–685.[Free Full Text]
  6. Garver, R. I., Jr., J.-F. Mornex, T. Nukiwa, M. Brantly, M. Courtney, J.-P. LeCoco, and R. G. Crystal. 1986. Alpha 1-antitrypsin deficiency and emphysema caused by homozygous inheritance of non-expressing alpha 1-antitrypsin genes. N. Engl. J. Med. 314:762–766.[Medline]
  7. Farber, J. L. 1994. Mechanisms of cell injury by activated oxygen species. Environ. Health Perspect. 102:17–24.
  8. Ogushi, F., R. C. Hubbard, C. Vogelmeier, G. A. Fells, and R. G. Crystal. 1991. Risk factors for emphysema: cigarette smoking is associated with a reduction in the association rate constant of lung {alpha}1-antitrypsin for neutrophil elastase. J. Clin. Invest. 87:1060–1065.
  9. Hayes, J. A., A. Korthy, and G. L. Snider. 1975. Pathology of elastase-induced panacinar emphysema in hamsters. J. Pathol. 117:1–14.[CrossRef][Medline]
  10. Kaplan, P. D., C. Kuhn, and J. A. Pierce. 1973. The induction of emphysema with elastase: I. The evolution of the lesion and the influence of serum. J. Lab. Clin. Med. 82:349–356.[Medline]
  11. Christensen, T. G., A. L. Korthy, G. L. Snider, and J. A. Hayes. 1977. Irreversible bronchial goblet cell metaplasia in hamsters with elastase-induced panacinar emphysema. J. Clin. Invest. 59:397–404.
  12. Furuno, T., T. Mitsuyama, K. Hidaka, T. Tanaka, and N. Hara. 1997. The role of neutrophil elastase in human pulmonary artery endothelial cell injury. Int. Arch. Allergy Immunol 112:262–269.[Medline]
  13. Venaille, T. J., G. Ryan, and B. W. Robinson. 1998. Epithelial cell damage is induced by neutrophil-derived, not pseudomonas-derived, proteases in cystic fibrosis sputum. Respir. Med. 92:233–240.[CrossRef][Medline]
  14. Kasahara, Y., R. M. Tuder, L. Taraseviciene-Stewart, T. D. Le Cras, S. Abman, P. K. Hirth, J. Waltenberger, and N. F. Voelkel. 2000. Inhibition of VEGF receptors causes lung cell apoptosis and emphysema. J. Clin. Invest. 106:1311–1319.[Medline]
  15. Massaro, G. D., and D. Massaro. 1997. Retinoic acid treatment abrogates elastase-induced pulmonary emphysema in rats. Nat. Med. 3:675–677.[CrossRef][Medline]
  16. Grummer, M. A., and R. D. Zachman. 1995. Postnatal rat lung retinoic acid receptor (RAR) mRNA expression and effects of dexamethasone on RAR-ß mRNA. Pediatr. Pulmonol. 20:234–240.[Medline]
  17. Jetten, A. M., A. R. Brody, M. A. Deas, G. E. R. Hook, J. I. Rearick, and S. M. Thacher. 1987. Retinoic acid and substratum regulate the differentiation of rabbit tracheal epithelial cells into squamous and secretory phenotype: morphological and biochemical characterization. Lab. Invest. 56:654–664.[Medline]
  18. Lechner, J. F., A. Haugen, I. A. McClendon, and E. W. Pettis. 1982. Clonal growth of normal adult human bronchial epithelial cells in a serum-free medium. In Vitro 18:633–642.[Medline]
  19. Ong, D. E., and F. Chytil. 1976. Changes in levels of cellular retinol– and retinoic acid–binding protein of liver and lung during perinatal development of rat. Proc. Natl. Acad. Sci. USA 73:3976–3978.[Abstract/Free Full Text]
  20. James, S. Y., M. A. Williams, A. C. Newland, and K. W. Colston. 1999. Leukemia cell differentiation: cellular and molecular interactions of retinoids and vitamin D. Gen. Pharmacol. 32:143–154.[CrossRef][Medline]
  21. Yang, J. J., R. Kettritz, R. J. Falk, J. C. Jennette, and M. L. Gaido. 1996. Apoptosis of endothelial cells induced by the neutrophil serine protease 3 and elastase. Am. J. Pathol. 149:1617–1626.[Abstract]
  22. Moreno-Manzano, V., Y. Ishikawa, J. Lucio-Cazana, and M. Kitamura. 1999. Supression of apoptosis by all-trans-retinoic acid: dual intervention in the c-Jun n-terminal kinase-AP-1 pathway. J. Biol. Chem. 274:20251–20258.[Abstract/Free Full Text]
  23. Yamaya, M., W. E. Finkbeiner, S. Y. Chun, and J. H. Widdicombe. 1992. Differentiated structure and function of cultures from human tracheal epithelium. Am. J. Physiol. 262:L713–L724.[Abstract/Free Full Text]
  24. Mosmann, T. 1983. Rapid colorimetric assay for cell growth and survival: application to proliferation and cytotoxicity assays. J. Immunol. Methods 65:55–63.[CrossRef][Medline]
  25. Fortenberry, J. D., M. L. Owens, and L. A. S. Brown. 1997. S-nitrosoglutathione enhances neutrophil DNA fragmentation and cell death. Am. J. Physiol. 276:L435–L442.
  26. Gavrieli, Y., Y. Sherman, and S. A. Ben-Sasson. 1992. Identification of programmed cell death in situ via specific lebeling of nuclear DNA fragmentation. J. Cell Biol. 119:493–501.[Abstract/Free Full Text]
  27. Fernandes-Alnemri, T., G. Litwack, and E. S. Alnemri. 1994. CPP32, a novel human apoptotic protein with homology to Caenorhabditis elegans cell death protein Ced-3 and mammalian interleukin-1ß-converting enzyme. J. Biol. Chem. 269:30761–30764.[Abstract/Free Full Text]
  28. Fujimoto, K., M. Ogawa, N. Saito, G. Kosaki, N. Minamura, and T. Yamamoto. 1980. A novel method of isolation and some characteristic properties of human pancreatic elastases. Biochim. Biophys. Acta. 612:262–267.[Medline]
  29. Nicholson, D. W., A. Ali, N. A. Thornberry, J. P. Vallancourt, C. K. Ding, M. Gallant, Y. Gareau, P. R. Griffin, M. Labelle, and Y. A. Lazebnik. 1995. Identification and inhibition of the ICE/CED-3 protease necessary for mammalian apoptosis. Nature 376:37–43.[CrossRef][Medline]
  30. Karlinsky, J. B., and G. L. Snider. 1978. Animal models of emphysema. Am. Rev. Respir. Dis. 117:1109–1133.[Medline]
  31. Kuhn, C., S. Yu, M. Chraplyvy, H. Linder, and R. M. Senior. 1976. The induction of emphysema with elastase: II. Changes in connective tissue. Lab. Invest. 34:372–380.[Medline]
  32. Nahori, M. A., P. Renesto, B. B. Vargaftig, and M. Chignard. 1992. Activation and damage of cultured airway epithelial cells by human elastase and cathepsin G. Eur. J. Pharmacol. 228:213–218.[Medline]
  33. Bardales, R. H., S. S. Xie, R. F. Schaefer, and S. M. Hsu. 1996. Apoptosis is a major pathway responsible for the resolution of type II pneumocytes in acute lung injury. Am. J. Pathol. 149:845–852.[Abstract]
  34. Tewari, M., L. T. Quan, K. O'Rourke, S. Desnoyers, Z. Zeng, D. R. Beidler, G. G. Poirier, G. S. Salvesen, and V. M. Dixit. 1995. Yama/CPP32ß, a mammalian homolog of CED-3, is a Crm-inhibitable protease that cleaves the death substrate poly (ADP-ribose) polymerase. Cell 81:801–808.[CrossRef][Medline]
  35. Torriglia, A., C. Negri, E. Chaudun, E. Prosperi, Y. Courtois, M. F. Counis, and A. I. Scovassi. 1999. Differential involvement of Dnases in HeLa cell apoptosis induced by etoposide and long term-culture. Cell Death Differ. 6:234–244.[CrossRef][Medline]
  36. Zakeri, Z. F., and H. S. Ahuja. 1997. Cell death/apoptosis: normal, chemically induced, and teratogenic effect. Mutat. Res. 396:149–161.[Medline]
  37. Kim, H. S., D. B. Hausman, M. M. Compton, R. G. Dean, R. J. Martin, G. J. Hausman, and D. L. Hartzell. 2000. Induction of apoptosis by all-trans-retinoic acid and C2-ceramide treatment in rat stromal-vascular cultures. Biochem. Biophys. Res. Commun. 270:76–80.[CrossRef][Medline]
  38. Taddeo, B., B. J. Nickoloff, and K. E. Foreman. 2000. Caspase inhibitor blocks human immunodeficiency virus 1-induced T-cell death without enhancement of HIV-1 replication and dimethyl sulfoxide increases HIV-1 replication without influencing T-cell survival. Arch. Pathol. Lab. Med. 124:240–245.[Medline]
  39. Sklan, D., P. Rappaport, and M. Vered. 1990. Inhibition of the activity of human leukocyte elastase by lipids particularly oleic acid and retinoic acid. Lung 168:323–332.[Medline]
  40. Trevani, A. S., G. Andonegui, M. Giordano, M. Nociari, P. Fontan, G. Dran, and J. R. Geffner. 1996. Neutrophil apoptosis induced by proteolytic enzymes. Lab. Invest. 74:711–721.[Medline]



This article has been cited by other articles:


Home page
 Am. J. Physiol. Lung Cell. Mol. Physiol.Home page
K. Ishizawa, T. Suzuki, M. Yamaya, Y. X. Jia, S. Kobayashi, S. Ida, H. Kubo, K. Sekizawa, and H. Sasaki
Erythromycin increases bactericidal activity of surface liquid in human airway epithelial cells
Am J Physiol Lung Cell Mol Physiol, October 1, 2005; 289(4): L565 - L573.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Respir. Crit. Care Med.Home page
S. Murakami, N. Nagaya, T. Itoh, T. Iwase, T. Fujisato, K. Nishioka, K. Hamada, K. Kangawa, and H. Kimura
Adrenomedullin Regenerates Alveoli and Vasculature in Elastase-induced Pulmonary Emphysema in Mice
Am. J. Respir. Crit. Care Med., September 1, 2005; 172(5): 581 - 589.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Lung Cell. Mol. Physiol.Home page
K. Takamura, Y. Nasuhara, M. Kobayashi, T. Betsuyaku, Y. Tanino, I. Kinoshita, E. Yamaguchi, S. Matsukura, R. P. Schleimer, and M. Nishimura
Retinoic acid inhibits interleukin-4-induced eotaxin production in a human bronchial epithelial cell line
Am J Physiol Lung Cell Mol Physiol, April 1, 2004; 286(4): L777 - L785.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Respir. Cell Mol. Bio.Home page
D. Massaro and G. D. C. Massaro
Retinoids, Alveolus Formation, and Alveolar Deficiency: Clinical Implications
Am. J. Respir. Cell Mol. Biol., March 1, 2003; 28(3): 271 - 274.
[Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Nakajoh, M.
Right arrow Articles by Sasaki, H.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Nakajoh, M.
Right arrow Articles by Sasaki, H.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Proc. Am. Thorac. Soc. Am. J. Respir. Crit. Care Med.
Copyright © 2003 American Thoracic Society.