Bleomycin-induced apoptosis of alveolar epithelial cells requires angiotensin synthesis de novo

Xiaopeng Li, Huiying Zhang, Valerie Soledad-Conrad, Jiaju Zhuang, and Bruce D. Uhal

Department of Physiology, Michigan State University, East Lansing, Michigan 48824


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Primary cultures of rat type II alveolar epithelial cells (AECs) or human AEC-derived A549 cells, when exposed to bleomycin (Bleo), exhibited concentration-dependent apoptosis detected by altered nuclear morphology, fragmentation of DNA, activation of caspase-3, and net cell loss over time. In both cell culture models, exposure to Bleo caused time-dependent increases in angiotensinogen (ANGEN) mRNA. Antisense oligonucleotides against ANGEN mRNA inhibited Bleo-induced apoptosis of rat AEC or A549 cells by 83 and 84%, respectively (P < 0.01 and P < 0.05), and prevented Bleo-induced net cell loss. Apoptosis of rat AECs or A549 cells in response to Bleo was inhibited 91% by the ANG-converting enzyme inhibitor captopril or 82%, respectively, by neutralizing antibodies specific for ANG II (both P < 0.01). Antagonists of ANG receptor AT1 (losartan, L-158809, or saralasin), but not an AT2-selective blocker (PD-123319), inhibited Bleo-induced apoptosis of either rat AECs (79%, P < 0.01) or A549 cells (83%, P < 0.01) and also reduced the activity of caspase-3 by 52% (P < 0.05). These data indicate that Bleo, like FasL or TNF-alpha , induces transactivation of ANG synthesis de novo that is required for AEC apoptosis. They also support the theory that ANG system antagonists have potential for the blockade of AEC apoptosis in situ.

renin-angiotensin system; type II pneumocyte; lung fibrosis; programmed cell death; angiotensinogen


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

ALVEOLAR EPITHELIAL CELLS (AECs) have many important roles that are critical to normal lung function (14). The death of AECs by apoptosis is now believed to be an important event in the pathogenesis of lung fibrosis (9) and in more acute lung injury (15, 24). A variety of investigations have implicated important roles for key molecules such as tumor necrosis factor-alpha (TNF-alpha ) and Fas ligand (FasL), both known inducers of apoptosis in a variety of cell types, in the events that lead to fibrogenesis in the lung (6, 7, 16).

In earlier work (28, 31), we showed that exposure of either primary cultures of rat AECs or the human AEC-derived A549 cell line to FasL or TNF-alpha increases angiotensinogen (ANGEN) mRNA and protein and evokes its subsequent conversion to angiotensin II (ANG II). Moreover, we found that transactivation of the ANGEN gene is required for AEC apoptosis in response to TNF-alpha or FasL. Thus AEC death in response to these agents could be blocked by ANG receptor antagonists or ANG-converting enzyme (ACE) inhibitors (ACEis), at least in vitro. Studies of another inducer of AEC apoptosis, the antiarrhythmic agent amiodarone, also showed that antagonists of ANG production or receptor interaction could prevent apoptosis of AECs in response to this benzofuran compound (1).

For these reasons we hypothesized that AEC apoptosis in response to bleomycin (Bleo) might also require ANG synthesis and might therefore be inhibitable by ANG system antagonists. We report here that Bleo, if applied to rat or human AECs in vitro, induces the expression of ANGEN mRNA and subsequent apoptosis that can be blocked by ANGEN antisense oligonucleotides, by ACEis, or by ANG receptor antagonists of the AT1-selective subtype.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Reagents and materials. The AT1-selective antagonists L-158809 and losartan were obtained from Merck (West Point, PA). The AT2-selective antagonist PD-123319 was obtained from Parke Davis Research Division (Ann Arbor, MI). The caspase inhibitor N-benzylcarboxy-Val-Ala-Asp-[O-Me]-CH2F (ZVAD-fmk) was obtained from Kamiya Biomedical (Seattle, WA). Asp-Glu-Val-Asp-[O-Me]-CH2F (DEVD-fmk) was obtained from Pharmingen (San Diego, CA). Alkaline phosphatase-conjugated streptavidin, digoxigenin-labeled deoxyuridine trisphosphate, and biotinylated deoxyuridine trisphosphate were obtained from Boehringer Mannheim (Indianapolis, IN). Bleo, anti-ANG antibodies, aurintricarboxylic acid (ATA), captopril, and saralasin were obtained from Sigma (St. Louis, MO). Reagents for detection of alkaline phosphatase and other secondary reagents for in situ end labeling of DNA or Western blotting were from sources described earlier (29). All other materials were of reagent grade and were obtained from Sigma.

Cell culture. The human lung adenocarcinoma cell line A549 was obtained from American Type Culture Collection and cultured in Ham's F-12 medium supplemented with 10% fetal bovine serum. Primary alveolar epithelial cells were isolated from adult male Wistar rats as described earlier (31). The primary cells were studied at day two of culture, a time at which they are type II cell-like by accepted morphological and biochemical criteria (17). Primary cell preparations were of >90% purity as assessed by acridine orange staining as described previously (25, 28, 31). All cells were grown in 24- or 6-well chambers and were analyzed at subconfluent densities of 80-90%. All subsequent incubations with Bleo and/or other test agents were performed in serum-free medium. The cells were exposed to caspase inhibitors or antagonists of the ANG system 30 min before exposure to Bleo for 1-20 h as indicated.

Quantitation of apoptosis and cell loss. Detection of apoptotic cells with propidium iodide (PI) was conducted as described earlier (28, 31) following digestion of ethanol-fixed cells with DNase-free RNase in PBS containing 5 µg/ml PI. In these assays, detached cells were retained by centrifugation of the 24-well culture vessels during fixation with 70% ethanol. Cells with discrete nuclear fragments containing condensed chromatin were scored as apoptotic. As in earlier publications, the induction of apoptosis was verified by in situ end labeling of fragmented DNA (see Fig. 1 and Refs. 29 and 126). Apoptotic cells were scored over a minimum of four separate microscopic fields from each of at least three culture vessels per treatment group.


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Fig. 1.   Detection of apoptosis in primary alveolar epithelial cells (AECs) and A549 cells. Primary cultures of AECs were exposed to vehicle (A) or bleomycin (Bleo, B and C) at 25 mU/ml for 20 h and were then fixed in 70% ethanol without washing (see MATERIALS AND METHODS). Cells exhibiting chromatin condensation and nuclear fragmentation with propidium iodide (PI; arrowheads, B) were scored as described earlier (Ref. 31 and MATERIALS AND METHODS). Under these conditions, only DNA appears red (27). C: Bleo-exposed cells were fixed and prepared for in situ end labeling (left) or terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (right) of fragmented DNA (29). Note colocalization of label (blue or brown, respectively) in nuclear fragments (arrowheads) identified by PI in B. A549 cells exposed to vehicle (D) or Bleo at 25 mU/ml (E) or 100 mU/ml (F) for 20 h were fixed and prepared for immunolabeling for the active form of caspase-3 (see MATERIALS AND METHODS). Note labeling of active caspase-3 (purple) in cells with either normal morphology (arrowheads, E) or in cells with condensed cytoplasm and nucleus (arrows, E and F). Note also reduced total cell number at increasing Bleo concentration (E and F).

Cell loss over 20 h of culture was quantitated by cell counts of the adherent plus detached cell populations. These were obtained following centrifugation of the culture vessels as described in the preceding paragraph, without prior washing. Thus detached cells (routinely <10% of the total cell number) were included in the cell loss data. Total cell counts (attached plus detached) were scored over a minimum of 200 nuclei per field, four fields per well with a minimum of six culture wells, or 4,800 nuclei, per treatment group. Data from each treatment group were compiled and analyzed by ANOVA followed by Student-Newman-Keuls post hoc analysis.

Detection and quantitation of caspase-3 activity. Activation of caspase-3 was detected through immunolabeling of fixed cells adherent to plastic culture surfaces with an antibody that recognizes only the active form of the enzyme (Biovision, Mountain View, CA). The primary antibody was detected with an alkaline phosphatase-conjugated secondary antibody followed by nitro-blue tetrazolium. The enzymatic activity of caspase-3 was measured in adherent cells incubated for 20 h with the membrane-permeable substrate N-acetyl-Asp-Glu-Val-Asp 7-amino-4-methylcoumarin (Upstate Biotech, Saranac Lake, NY) at 50 µM. Quantitation of the fluorescent product was achieved with a Biotek FL600 fluorescence plate reader. Fluorescence values were normalized to cell number determined on the same culture well after cell fixing and staining of DNA with PI (27).

RT-PCR and antisense experiments. Semiquantitative RT-PCR was performed as described earlier (28, 31). The annealing temperatures for PCR reactions were optimized for each primer by preliminary trials. All PCR amplifications were terminated at or near the center of the linear range for each gene product analyzed, as determined by sequential withdrawal of sample at five-cycle intervals between 20 and 40 cycles (not shown). The identity of expressed genes was determined by expected size of the PCR product in 1.6% agarose gels.

For RT-PCR of rat-specific gene products, the following primers were used: for ANGEN, coding = 5'-CCTCGCTCTCTGGACTTATC-3' and uncoding = 5'-CAGACACTGAGGTGCTGTTG-3', which yields a PCR product of 226 bp by single-step RT-PCR (20). For beta -microglobulin, the primers used were: coding = 5'-CTCCCCAAA-TTCAAGTGTACTCTCG-3' and uncoding = 5'-GAGTGACGTGTTTAACTCTGCA-AGC-3', which yields a product of 249 bp (11). For RT-PCR from human A549 cells, the following primers were used: for ANGEN, coding = 5'-GCTTTC-AACACCTACGTCCA-3' and uncoding = 5'-AGCTGTTGGGTAGACTCTGT-3'. These primers yield a final PCR product of 509 bp (12). For beta -actin, single-step RT-PCR was used with the primers: coding = 5'-AGG-CCAACCGCGAGAAGATGACC-3' and uncoding = 5'-GAAGTCCAGGGCGACGT-AGC-3', which produces a PCR product of 332 bp (21).

For antisense studies, phosphorothioated control and antisense oligonucleotides against ANGEN (18-mers) were synthesized and transfected into A549 cells or primary rat AEC (both at 40 nM final concentration) using the lipofectin reagent OligofectAMINE (Invitrogen Life Technologies, Grand Island, NY) at 6 µl/ml as the vehicle, diluted in the OPTIMEM medium accompanying the lipofectin. The control nucleotides were of the same length and base composition as the antisense, but with scrambled sequence. The oligonucleotide-lipofectin ratio was optimized (over a 4-h transfection) to yield transfection efficiencies of 50-75% with no apparent cell loss or detachment. Transfection efficiency was monitored with FITC-labeled 25-mer oligonucleotide for luciferase (not shown). Transfections were conducted for 4 h followed by 5× washing with serum-free cell culture medium; immediately thereafter, Bleo or vehicle was applied as described in Cell culture for 20 h. The transfection protocol itself had no significant effect on basal or Bleo-induced apoptosis (see RESULTS). Phosphorothioated oligonucleotides used for transfection were: (ANGEN antisense) 5-CCGTGGGAGTCATCACGG-3' and (ANGEN scramble) 5'-CAGGGATCTCTGGCGGAC-3' as described by Phillips et al. (19).


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Exposure of primary cultures of rat AECs or A549 cells to Bleo for 20 h caused apoptosis detectable by nuclear fragmentation, DNA fragmentation, and immunolabeling of the active form of caspase-3 (Fig. 1). Although these markers were detected in a minor fraction of the cells, the apoptosis induced was sufficient to reduce the total cell number significantly over time (see Fig. 1, D-F, and quantitation in Fig. 7B). Scoring of fragmented nuclei revealed dose-dependent apoptosis that reached statistical significance beginning at 0.5 mU/ml in primary AECs (Fig. 2) and at 1 mU/ml in A549 cells (not shown). The nuclear fragmentation was blocked by the broad-spectrum caspase inhibitor ZVAD-fmk (60 µM) or by the endonuclease inhibitor ATA (10 µM), confirming the specificity of the assay for apoptosis. Apoptosis of the rat AECs also was blocked by the ACE inhibitor captopril (500 ng/ml) and by the nonselective ANG receptor antagonist saralasin (50 µg/ml), in agreement with earlier studies of Fas- and TNF-induced AEC apoptosis (28, 31).


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Fig. 2.   Dose-dependent induction of nuclear fragmentation by Bleo in primary rat AECs and blockade by inhibitors of caspases, endonucleases, ANG-converting enzyme (ACE), and ANG-receptor interaction. Rat AECs were isolated and challenged with the indicated concentrations of Bleo on day 2 of primary culture (see MATERIALS AND METHODS). Putative inhibitors were added 30 min before addition of Bleo; nuclear fragmentation was scored as described in Fig. 1B and MATERIALS AND METHODS. ZVAD, N-benzylcarboxy-Val-Ala-Asp-[O-Me]-CH2F (60 µM); ATA, aurintricarboxylic acid (10 µM); Capto, captopril (500 ng/ml); Saral, saralasin (50 µg/ml). Bars are the means ± SE of at least 4 observations; *P < 0.05 vs. control (0.0 Bleo).

In a separate experiment (Fig. 3), Bleo-induced apoptosis of primary AECs was blocked by the caspase-3-selective blocker DEVD-fmk (60 µM) and by the ANG receptor subtype AT1-selective blocker losartan (10-6 M, P < 0.01), suggesting that subtype AT1 mediates Bleo-induced apoptosis as it does AEC apoptosis in response to ANG II (18). This was found to be the case in human AECs as well (Fig. 4); Bleo-induced apoptosis of A549 cells was inhibited 83% by the AT1-selective blocker L-158809 but was not reduced by the AT2-selective antagonist PD-123319. Moreover, Bleo-induced apoptosis was also prevented by a neutralizing antibody specific for ANG II, but not by an isotype-matched nonimmune immunoglobulin. Furthermore, the total enzymatic activity of caspase-3 was elevated by exposure of A549 cells to Bleo (Fig. 5), but the increase was inhibited 52% by saralasin.


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Fig. 3.   Blockade of Bleo-induced apoptosis in primary AECs by selective caspase or ANG receptor blockers. Rat AECs were isolated and challenged with 25 mU/ml Bleo alone or in the presence of the caspase-3-selective inhibitor Asp-Glu-Val-Asp-[O-Me]-CH2F (DEVD-fmk, 60 µM) or the ANG receptor AT1-selective antagonist losartan (Los, 10-6 M). Control cultures (Ctl) received blocker vehicles only. Nuclear fragmentation was scored as described in Fig. 1 and MATERIALS AND METHODS. Bars are means ± SE of at least 4 observations; *P < 0.05 vs. control.



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Fig. 4.   Inhibition of Bleo-induced apoptosis of A549 cells by inhibitors of caspases or ANG II-receptor interaction. A549 cells were cultured to 80% confluence as described in MATERIALS AND METHODS and were challenged with 25 mU/ml Bleo in the presence or absence of the indicated compounds. Anti-ANG II, neutralizing antibody to ANG II (1 µg/ml); NS IgG, isotype-matched nonimmune immunoglobulin (1 µg/ml); L-158809, ANG receptor AT1-selective antagonist (10-6 M); PD-123319, ANG receptor AT2-selective antagonist (10-6 M). Other abbreviations and concentrations are the same as in Figs. 2 and 3. Nuclear fragmentation was scored as described in Fig. 1 and MATERIALS AND METHODS. Bars are means ± SE of at least 4 observations; *P < 0.05 vs. control (0 dose).



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Fig. 5.   Induction of caspase-3 activity by Bleo and inhibition by an ANG receptor antagonist. A549 cells were challenged with Bleo (25 mU/ml) for 20 h in the presence and absence of the nonselective ANG receptor antagonist Saral (50 µg/ml). Assay of caspase-3 was conducted on adherent cells as described in MATERIALS AND METHODS. *P < 0.05 vs. control; **P < 0.05 vs. Bleo.

These data suggested that Bleo induces ANG II synthesis de novo in primary AECs and A549 cells. Consistent with this theory, semiquantitative RT-PCR for ANGEN revealed more abundant ANGEN mRNA in primary AECs at 3 h and especially at 20 h after challenge with 25 mU/ml Bleo (Fig. 6A). In A549 cells (Fig. 6B), 25 mU/ml Bleo stimulated a significant increase in ANGEN mRNA that was detectable at 1 h after addition of Bleo and increased by 7 h.


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Fig. 6.   Semiquantitative RT-PCR of angiotensinogen (ANGEN) mRNA in AECs after Bleo exposure. Primary cultures of rat AECs (A) and A549 cells (B) were exposed to Bleo (25 mU/ml) for the indicated times, and total RNA was isolated. RT-PCR was performed as described before (28, 31) with primers specific for rat or human ANGEN, beta -microglobulin (beta -MG), or beta -actin as control mRNAs.

To determine whether functional ANGEN mRNA is required for the apoptotic response to Bleo, we transfected phosphorothioated antisense or scrambled-sequence control oligonucleotides against ANGEN mRNA into rat AECs and A549 cells immediately before challenge with Bleo for an additional 20 h. As shown in Fig. 7A, Bleo-induced apoptosis of primary AECs was inhibited by 83% by the ANGEN antisense but not by the scrambled control oligonucleotides. In A549 cells (Fig. 7B), the antisense also reduced Bleo-induced apoptosis by 84%, but the scrambled oligonucleotide had no significant effect. Moreover, exposure to Bleo for 20 h reduced the total cell number of A549 cells (Fig 7B, bottom) by 43%, but the ANGEN antisense prevented the Bleo-induced cell loss.


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Fig. 7.   Blockade of bleomycin (Ble) induced apoptosis and net cell loss in AECs and A549 cells by antisense oligonucleotides against ANGEN mRNA. Primary rat AECs (A) or A549 cells (B) were transfected for 4 h with antisense or scrambled sequence (scram) oligonucleotides as described earlier (Ref. 31 and MATERIALS AND METHODS). Cells were challenged with Ble (25 mU/ml) for 20 h immediately thereafter, and apoptosis (top) was scored as detailed in Figs. 1-3; net cell loss (B, bottom) was measured as detailed in MATERIALS AND METHODS. lipo, Lipofectamine; see MATERIALS AND METHODS for details. Bars are means ± SE of at least 4 observations; *P < 0.05; **P < 0.01 vs. corresponding control.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The results described here agree with previous studies from this laboratory that showed a requirement for autocrine ANG II production and receptor interaction for AEC apoptosis in response to FasL or TNF-alpha (28, 31). Other authors also have reported DNA damage and death of AECs in response to Bleo (7, 10). Although a previous report of Bleo action on A549 cells described no influence of the drug on cell viability in vitro (22), the levels of apoptosis described here (apoptotic index ~10-20%, see Figs. 1 and 2) suggest that cell death at the relatively low doses used in that study (0.1-2 mU/ml) may have gone undetected. Nonetheless, the levels of apoptosis reported here are more than sufficient to result in significant net cell loss in a relatively short period of time (see Fig. 7). More importantly, both the cell loss and markers of apoptosis could be blocked by ANG system antagonists, consistent with the theory that these agents can prevent apoptosis, and thus cell loss, in response to Bleo.

The findings that Bleo-induced apoptosis of AECs could be blocked by the AT1-selective antagonists losartan (Fig. 3) and L-158809 (Fig. 4) but not by the AT2-selective antagonist PD-123319 are in agreement with our recent demonstration that the AT1 receptor subtype mediates AEC apoptosis in response to purified ANG II (18). They also support the contention that autocrine production of ANG II and binding to its receptor(s) are required for the apoptotic response. This contention is also supported by the ability of ANGEN antisense oligonucleotides or a neutralizing antibody that recognizes ANG II, but not ANG I or ANGEN, to essentially abrogate apoptosis and prevent net cell loss in response to bleomycin (Figs. 4 and 7).

In an earlier report we described the induction of apoptosis in AECs by exposure to purified ANG II (32), which also occurred in a concentration-dependent manner with an EC50 of 10 and 50 nM for primary AECs and A549 cells, respectively. Those data indicated that exposure of AECs to exogenous ANG II is sufficient, in the absence of other stimuli, for the induction of apoptosis. Measurements of the ANG II concentration in the cell culture media at a single sampling time (20 h) suggest that Bleo increases ANG II in the medium by at least two- to threefold, to a level close to that required for induction of apoptosis by purified ANG II (2-3 nM, data not shown). However, intracellular receptors for ANG II have been demonstrated in other cell types (4, 8), and ANG II administered intracellularly by microinjection (8) has been shown to invoke a variety of signaling pathways. Thus, in the present study, intracellular generation of ANG II by Bleo may be sufficient to stimulate apoptosis independently of extracellular ANG II. In both serum and interstitial fluid, the half-life of ANG II is short (15 s-15 min), and receptor-bound ANG II is known to be internalized in many cell types (5); for all these reasons, the interpretation of extracellular ANG II levels is difficult at best.

The RT-PCR data of Fig. 7 and measurements of ANGEN protein by Western blotting (not shown) suggest that there is a basal level of ANGEN expression by AECs, even in the absence of other proapoptotic stimuli. In experiments not reported here, the ANG receptor antagonist saralasin, applied alone, decreased the basal rate of spontaneous AEC apoptosis (normally 1-2% of total cells at steady state); this finding supports the notion that basal ANGEN expression is involved in the basal rate of AEC death. On the other hand, the rate at which ANGEN protein is proteolytically cleaved, both inside and outside the cell, likely constitutes another point of regulation, but at present we have little data to indicate which point of control is most critical. Moreover, it seems reasonable to suspect that Bleo and other inducers of AEC apoptosis alter the expression of additional components of the local renin-angiotensin system in AECs, such as angiotensin-converting enzymes and receptors as well as ANGEN expression. Consistent with this theory, recent work by Day et al. (3) demonstrated upregulation of ACE in cultured endothelial cells by Bleo. Alveolar epithelial cells also express ACE mRNA, and ACE inhibitors block AEC apoptosis in response to FasL (31), TNF-alpha (28), and Bleo (Fig. 2). Investigations to define the possible regulation of ACE and other peptidases in AECs are currently underway.

Although the mechanism by which Bleo upregulates ANGEN mRNA was not addressed in this study, the findings that Bleo, FasL, and TNF-alpha all increased ANGEN mRNA (28, 31) imply the involvement of a pathway common to these inducers. Regulation of ANGEN expression is best studied in the hepatocyte, in which the stimulatory effects of TNF-alpha and interleukin-6 have been shown to be mediated through the interaction of the transcription factors NF-kappa B and STAT3 with the acute phase response element of the ANGEN promoter (2, 23). In contrast, studies of the regulation of ANGEN expression by the cardiac myocyte have shown that p53 is a key regulator of its expression in response to a variety of stimuli (13, 20). Whether this difference reflects the distinct developmental lineages of these cell types or other factors is unknown. Viewed in this context, future investigations of the regulation of ANGEN expression by cells of the lung will be most interesting, particularly in light of the fact that the lung contains many different cell types that are in very close proximity but arose from distinct embryologic origins. As an example, the results reported here for AECs complement our earlier report of ANGEN expression by human lung myofibroblasts (30), which reside immediately adjacent to AECs in fibrotic lung in situ.

In summary, exposure of primary cultures of rat alveolar epithelial cells or the AEC-derived A549 cell line to Bleo caused a time-dependent increase in ANGEN mRNA and caspase-dependent apoptosis. In either cell type, the apoptosis could be prevented by agents that prevent the synthesis of ANGEN protein, its conversion to the mature peptide ANG II, or the binding of the peptide to its receptors. These findings may have important implications toward possible therapeutic strategies to prevent lung injury and/or fibrogenesis. They also raise the possibility that previous clinical trials involving ACEis or ANG receptor antagonists may hold useful information related to the potential of these agents to affect pulmonary disease.


    ACKNOWLEDGEMENTS

This work was supported by National Heart, Lung, and Blood Institute Public Health Service Grant HL-45136, American Heart Association Grant-In-Aid 0250269N, and by the Michigan State University Foundation.


    FOOTNOTES

Address for reprint requests and other correspondence: B. D. Uhal, Dept. of Physiology, Michigan St. Univ., 3185 Biomedical and Physical Sciences Bldg., E. Lansing, MI 48824 (E-mail: uhal{at}msu.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

First published November 15, 2002;10.1152/ajplung.00273.2002

Received 8 August 2002; accepted in final form 5 November 2002.


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ABSTRACT
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MATERIALS AND METHODS
RESULTS
DISCUSSION
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26.   Uhal, BD, Joshi I, Ramos C, Pardo A, and Selman M. Alveolar epithelial cell death adjacent to underlying myofibroblasts in advanced fibrotic human lung. Am J Physiol Lung Cell Mol Physiol 275: L1192-L1199, 1998[Abstract/Free Full Text].

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28.   Wang, R, Alam G, Zagariya A, Gidea C, Pinillos H, Lalude O, Choudhary G, and Uhal BD. Apoptosis of lung epithelial cells in response to TNF-alpha requires angiotensin II generation de novo. J Cell Physiol 185: 253-259, 2000[ISI][Medline].

29.   Wang, R, Ibarra-Sunga O, Pick R, and Uhal BD. Abrogation of bleomycin-induced epithelial apoptosis and lung fibrosis by captopril or by a caspase inhibitor. Am J Physiol Lung Cell Mol Physiol 279: L143-L151, 2000[Abstract/Free Full Text].

30.   Wang, R, Ramos C, Joshi I, Zagariya A, Ibarra-Sunga O, Pardo A, Selman M, and Uhal BD. Human lung myofibroblast-derived inducers of alveolar epithelial apoptosis identified as angiotensin peptides. Am J Physiol Lung Cell Mol Physiol 277: L1158-L1164, 1999[Abstract/Free Full Text].

31.   Wang, R, Zagariya A, Ang E, Ibarra-Sunga O, and Uhal BD. Fas-induced apoptosis of alveolar epithelial cells requires angiotensin II generation and receptor interaction. Am J Physiol Lung Cell Mol Physiol 277: L1245-L1250, 1999[Abstract/Free Full Text].

32.   Wang, R, Zagariya A, Ibarra-Sunga O, Gidea C, Ang E, Deshmukh S, Chaudhary G, Baraboutis J, Filippatos G, and Uhal BD. Angiotensin II induces apoptosis in human and rat alveolar epithelial cells. Am J Physiol Lung Cell Mol Physiol 276: L885-L889, 1999[Abstract/Free Full Text].


Am J Physiol Lung Cell Mol Physiol 284(3):L501-L507
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