RAPID COMMUNICATION
Fas-induced apoptosis of alveolar epithelial cells requires ANG II generation and receptor interaction

Rongqi Wang1, Alex Zagariya2, Edmund Ang1, Olivia Ibarra-Sunga1, and Bruce D. Uhal1

1 The Cardiovascular Institute and 2 Division of Neonatology, Michael Reese Hospital and Medical Center, Chicago, Illinois 60616


    ABSTRACT
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ABSTRACT
INTRODUCTION
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REFERENCES

Recent works from this laboratory demonstrated potent inhibition of Fas-induced apoptosis in alveolar epithelial cells (AECs) by the angiotensin-converting enzyme (ACE) inhibitor captopril [B. D. Uhal, C. Gidea, R. Bargout, A. Bifero, O. Ibarra-Sunga, M. Papp, K. Flynn, and G. Filippatos. Am. J. Physiol. 275 (Lung Cell. Mol. Physiol. 19): L1013-L1017, 1998] and induction of dose-dependent apoptosis in AECs by purified angiotensin (ANG) II [R. Wang, A. Zagariya, O. Ibarra-Sunga, C. Gidea, E. Ang, S. Deshmukh, G. Chaudhary, J. Baraboutis, G. Filippatos and B. D. Uhal. Am. J. Physiol. 276 (Lung Cell. Mol. Physiol. 20): L885-L889, 1999]. These findings led us to hypothesize that the synthesis and binding of ANG II to its receptor might be involved in the induction of AEC apoptosis by Fas. Apoptosis was induced in the AEC-derived human lung carcinoma cell line A549 or in primary AECs isolated from adult rats with receptor-activating anti-Fas antibodies or purified recombinant Fas ligand, respectively. Apoptosis in response to either Fas activator was inhibited in a dose-dependent manner by the nonthiol ACE inhibitor lisinopril or the nonselective ANG II receptor antagonist saralasin, with maximal inhibitions of 82 and 93% at doses of 0.5 and 5 µg/ml, respectively. In both cell types, activation of Fas caused a significant increase in the abundance of mRNA for angiotensinogen (ANGEN) that was unaffected by saralasin. Transfection with antisense oligonucleotides against ANGEN mRNA inhibited the subsequent induction of Fas-stimulated apoptosis by 70% in A549 cells and 87% in primary AECs (both P < 0.01). Activation of Fas increased the concentration of ANG II in the serum-free extracellular medium 3-fold in primary AECs and 10-fold in A549 cells. Apoptosis in response to either Fas activator was completely abrogated by neutralizing antibodies specific for ANG II (P < 0.01), but isotype-matched nonimmune immunoglobulins had no significant effect. These data indicate that the induction of AEC apoptosis by Fas requires a functional renin-angiotensin system in the target cell. They also suggest that therapeutic control of AEC apoptosis is feasible through pharmacological manipulation of the local renin-angiotensin system.

programmed cell death; renin-angiotensin system; type II pneumocyte; pulmonary fibrosis; angiotensin-converting enzyme


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
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DISCUSSION
REFERENCES

PULMONARY ALVEOLAR EPITHELIAL CELLS (AECs) have varied and important roles in lung homeostasis, one of which is the replacement of cells lost to lung injury (11). The rate of AEC death is a critical factor determining the capacity of the epithelium to repair damage (18), and the large fraction of that rate composed of apoptotic cell death is only recently being realized (2, 7). A growing body of evidence suggests that Fas-induced apoptosis of AECs is an important factor in the pathogenesis of fibrotic lung diseases (4, 5).

The onset of experimental lung fibrosis has long been known to be inhibited by antagonists of the renin-angiotensin system (12, 24, 25). Earlier work from this laboratory (20) showed potent inhibition of Fas-induced apoptosis of a human lung epithelial cell line by captopril, an angiotensin (ANG)-converting enzyme (ACE) inhibitor already known to block lung fibrogenesis (12). This finding suggested that the conversion of ANG I to ANG II might be involved in Fas-induced apoptosis by AECs. That theory was made plausible by the recent demonstration by Wang et al. (23) that purified ANG II induces dose-dependent apoptosis of cultured AECs that is inhibitable by ANG II receptor antagonists.

The same study found that AECs in vitro constitutively express the mRNAs for both subtypes of ANG II receptor as well as for ACE. Furthermore, primary AECs exposed to purified angiotensinogen (ANGEN) underwent apoptosis that was both ANG II receptor dependent and inhibitable by ACE inhibitors. Those data indicated that cultured AECs express all renin-ANG system components necessary for the enzymatic conversion of ANGEN to ANG II and the subsequent induction of apoptosis. This study examines the possibility that the synthesis of ANG II de novo and its subsequent binding to the ANG II receptor might be necessary for apoptosis in response to Fas. We report here that the induction of ANGEN expression, its proteolytic processing, and the subsequent binding of ANG II to its receptor are required events in the signaling of AEC apoptosis by Fas.


    METHODS
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INTRODUCTION
METHODS
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REFERENCES

Reagents and materials. Purified ANG II, ANGEN, lisinopril, saralasin, and antibodies to ANG II and ANGEN were obtained from Sigma (St. Louis, MO). Primers for RT-PCR were synthesized by Genemed Synthesis (San Francisco, CA). Lipofectin reagent (Oligofectin G) was obtained from Sequitur (Natick, MA). All other materials were from sources described earlier (20, 23) or were of reagent grade.

Cell culture and detection of apoptosis. 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 AECs were isolated from adult male Wistar rats as described earlier (21, 23). The primary cells were studied on day 2 of culture, a time at which they are type II cell-like by accepted morphological and biochemical criteria (18), and all preparations were of >90% purity as assessed by acridine orange staining as previously described (19). All cells were seeded in 24-well or 6-well chambers, and all experiments were conducted at subconfluent densities of 80-90% in serum-free Ham's F-12 medium. Test reagents were diluted with serum-free Ham's F-12 medium and were applied under serum-free conditions for 20 h at 37°C in a 5% CO2 incubator. Detection of apoptotic cells with propidium iodide was conducted as described earlier (20, 21, 23) after digestion of ethanol-fixed cells with DNase-free RNase in PBS containing 5 µg/ml of propidium iodide. In all assays, detached cells were retained by centrifugation of the culture vessels during fixation with 70% ethanol or by retention of the culture medium and recovery by centrifugation before the assay. As in earlier publications (22, 23), the induction of apoptosis was verified by annexin V binding and generation of DNA strand breaks.

RT-PCR. RT-PCR assay of ANGEN gene products was performed essentially as described earlier (23). Briefly, total RNA was isolated with the RNeasy Mini protocol (Qiagen, Santa Clarita, CA). To synthesize cDNA by RT-PCR, 3 µg of purified RNA were reverse transcribed with 2 µM oligo(dT), 50 mM Tris · HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 0.01 mM dithiothreitol, 0.2 mM each deoxynucleotide triphosphate, 1 U/µl of RNase inhibitor (RNasin), and 2 U of avian myoblastosis virus reverse transcriptase (Promega, Madison, WI) in a total volume of 30 µl. The RT reaction was performed for 1 h at 42°C followed by 20 cycles of PCR amplification, which was at or near the center of the linear range for each gene product analyzed (data not shown).

PCR amplification was performed with 10-µl aliquots of cDNA obtained as described above, equivalent to 1 µg of the total starting RNA extract. The amplification reaction was performed in 50 µl of PCR buffer containing 5 mM MgCl2, 5 µg/ml of each 5' and 3' primer (see below), 2 µl of 10 mM deoxynucleotide triphosphates, and 1 U of Taq polymerase (Promega) with a Perkin-Elmer PCR amplifier. The PCRs included denaturation at 95°C for 30 s, annealing of primers for 30 s, and elongation of the chain at 72°C for 1 min with Taq DNA polymerase. The annealing temperatures for RT-PCRs were optimized for each primer by preliminary trials. For any given primer, the control and treated samples were multiplexed within the same RT-PCR. Samples were stored at 4°C; negative controls lacked DNA. Identity of the expressed genes was determined by the expected size of the PCR product on 1.6% agarose gels.

For RT-PCR from the human A549 cells, the primers used for ANGEN were an "outer" primer of a two-step nested assay: coding, 5'-GCTTTCAACACCTACGTCCA-3', and uncoding, 5'-AGCTGTTGGGTAGACTCTGT-3', and an "inner" primer of the nested assay: coding, 5'-TTCTCCCTGCTGGCCGAG-3', and uncoding, 5'-GGGCTCTCTCTCATCCGC-3'. These primers yielded a final PCR product of 447 bp (8). For beta -actin, single-step RT-PCR was used with the primers coding, 5'-AGGCCAACCGCGAGAAGATGACC-3', and uncoding, 5'-GAAGTCCAGGGCGACGTAGC-3', which produced a PCR product of 332 bp (17).

For RT-PCR of rat-specific gene products, the primers used for ANGEN were coding, 5'-CCTCGCTCTCTGGACTTATC-3', and uncoding, 5'-CAGACACTGAGGTGCTGTTG-3', which yielded a PCR product of 226 bp by single-step RT-PCR (16). For beta -microglobulin, the primers used were coding, 5'-CTCCCCAAATTCAAGTGTACTCTCG-3', and uncoding, 5'-GAGTGACGTGTTTAACTCTGCAAGC-3', which yielded a product of 249 bp (6).

Antisense transfection and in situ hybridization. Phosphorothioated control and antisense oligonucleotides against ANGEN (18 mers) were synthesized and transfected into A549 cells or primary rat AECs with the lipofectin reagent Oligofectin G (Sequitur) as the vehicle diluted in cell culture medium. The control nucleotides were of the same length and base composition as the antisense but with a scrambled sequence. The oligonucleotide-to-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 a FITC-labeled 25-mer oligonucleotide for luciferase (data not shown). Transfections were conducted for 4 h followed by five washes with serum-free cell culture medium; immediately thereafter, Fas activator or vehicle was applied as described above for 20 h. The transfection protocol itself had no significant effect on basal or Fas-induced apoptosis (data not shown). Phosphorothioated oligonucleotides used for transfection were ANGEN antisense, 5'-CCGTGGGAGTCATCACGG-3', and ANGEN scramble, 5'-CAGGGATCTCTGGCGGAC-3', as described by Phillips et al. (15).

In situ hybridization was performed essentially as described by Panoskaltsis-Mortari and Bucy (14) except that no 80°C denaturation was employed because single-stranded probes were used. Fixed A549 cells were hybridized with digoxigenin-labeled antisense and control oligonucleotide DNA probes specific for ANGEN, which were detected with an amplified biotin-avidin system linked to a nitro blue tetrazolium chromogen (purple is positive). As in the transfection experiments, the control probes were of the same length and base composition as the antisense probe but with a scrambled sequence. The digoxigenin-labeled probes used were antisense, 5'-AGGGTGGGGGAGGTGCTGAACAGC-3', and scramble, 5'-GATGGGGGTGGGGGACCGTAGCAA-3', as described by Lai et al. (8).


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Fas-induced apoptosis of A549 cells was significantly inhibited by the nonthiol ACE inhibitor lisinopril (Fig. 1A) and was essentially abrogated by the nonselective ANG II receptor antagonist saralasin (maximal inhibition of 93% at 50 µg/ml; Fig. 1B). In agreement with the data from A549 cells, apoptosis within primary cultures of rat AECs (induced by purified recombinant Fas ligand) was also blocked by saralasin (Fig. 1C) or by captopril or lisinopril, both at 500 ng/ml (Fig. 1D).


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Fig. 1.   Abrogation of Fas-induced apoptosis in cultured alveolar epithelial cells (AECs) by angiotensin-converting enzyme (ACE) inhibitors or an angiotensin II receptor antagonist. A human lung epithelial cell line (A549 EPITH; A and B) or primary isolates of rat AECs (C and D) were exposed to receptor-activating anti-Fas antibody (clone CH-11) or recombinant human Fas ligand (FASL), respectively, for 20 h (see METHODS and Ref. 19). Apoptosis was detected by nuclear fragmentation as described earlier (19, 23). Data are percent inhibition of Fas-induced apoptosis in response to indicated concentrations of lisinopril (LISIN; A) or saralasin (B and C) or a single concentration of 500 ng/ml of captopril (CAPTO) or LISIN (D). Data are means ± SE; n = 3 or more cultures in each of 2 separate experiments. Significantly different from no inhibitor: * P < 0.05; ** P < 0.01 (both by ANOVA and Dunnett's test).

These findings suggested that Fas-induced apoptosis of AECs might be dependent on de novo synthesis of ANG II and its binding to one of its receptors. Consistent with this model, quantitative RT-PCR (Fig. 2) revealed that activation of Fas in either A549 cells (A) or primary rat AECs (B) causes significant increases in the abundance of mRNA for ANGEN but not in the control transcripts beta -actin and beta -microglobulin. In both cell types, the presence of saralasin during Fas activation had no effect on the accumulation of ANGEN mRNA.


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Fig. 2.   RT-PCR analysis of angiotensinogen (ANGEN) mRNA abundance in response to Fas activation in AECs. Total RNA was isolated from human A549 cells (A) or primary rat AECs (B) that were unactivated (CTL) or activated with Fas-ligating CH-11 antibody or purified FASL in presence and absence of saralasin (SAR). Quantitative RT-PCR (see METHODS) was performed with primers specific for human or rat ANGEN, human beta -actin, or rat beta -microglobulin (beta -MG). CTL, control. Arrows, molecular-size standards (STD) in bp. Note increase in ANGEN RT-PCR product in response to Fas activation in both cell types irrespective of SAR.

In Fig. 3, a 4-h transient transfection of antisense oligonucleotides against ANGEN significantly inhibited Fas-induced apoptosis of either A549 cells (left) or primary rat AECs (right) over the subsequent 20 h posttransfection (70 and 87% inhibition, respectively; both P < 0.01 vs. Fas plus lipofectin). In contrast, transfection of control oligonucleotides of the same size but with a scrambled sequence produced no significant inhibition in A549 cells and only 28% inhibition in primary AECs (P < 0.05). The LipofectAMINE transfection itself had no significant effect on either basal or Fas-induced apoptosis (data not shown). When related antisense oligonucleotides against ANGEN (see METHODS) were used for in situ hybridization (Fig. 4), an intense positive signal (purple) was found within cells that contained the fragmented nuclei typical of apoptotic cells (arrowheads). No positive signal was found in cells hybridized with control oligonucleotides.


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Fig. 3.   Inhibition of Fas-induced apoptosis by antisense oligonucleotides against ANGEN mRNA. Human A549 cells (left) and primary rat AECs (right) were exposed for 4 h to lipofectin (LIPO) transfection reagent in presence and absence of antisense oligonucleotides to ANGEN (ANTISENSE) or control oligonucleotides of the same length and base composition but with scrambled sequence (SCRAMBLE). Cells were then washed, and over the subsequent 20 h, apoptosis was induced (FAS) by activating anti-Fas antibodies (A549 cells) or purified FASL (primary AECs) and was detected as described in Fig. 1. Note significant blockade by prior exposure to ANTISENSE but not by SCRAMBLE. Values are means ± SE; n = 3 or more in each of 2 separate experiments. Significantly different (P < 0.01) from: * CTL; ** FAS+LIPO (both by ANOVA and Student-Newman-Keuls test).



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Fig. 4.   Detection of ANGEN mRNA in A549 cells by in situ hybridization. Human A549 cells were incubated with activating anti-Fas antibody (+FAS) as in Fig. 1 and were fixed for optimal retention of RNA (see METHODS). Nonisotopic in situ hybridization was performed with oligonucleotides specific for ANGEN mRNA (ANGEN ANTISENSE) or control oligonucleotides of the same length and composition but with a scrambled sequence (ANGEN SCRAMBLE). Positive reaction is purple. Note intense signal in cells with fragmented nuclei (arrowheads), but lack of signal in cells with normal nuclear morphology (arrow) and in cells exposed to SCRAMBLE. See METHODS for details.

Enzyme-linked immunosorbent assay specific for ANG II (Fig. 5) revealed that activation of Fas significantly increased the concentration of free ANG II peptide in the culture medium bathing primary AECs (left) or A549 cells (right). Figure 6 shows that Fas-induced apoptosis of either rat AECs (top) or A549 cells (bottom) could be completely abrogated by neutralizing antibodies specific for ANG II. The specificity of this experimental approach is supported by the finding that the same antibody completely blocked AEC apoptosis induced by purified ANG II, whereas isotype-matched nonimmune IgGs had no significant effects. The reliability of the nuclear fragmentation assay as an index of apoptosis is shown by the ability of the broad-spectrum caspase inhibitor Z-Val-Ala-Asp-fluoromethylketone (20) to abrogate the effect of Fas ligand (Fig. 6, top).


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Fig. 5.   Effect of Fas activation on extracellular angiotensin (ANG) II generated by AECs. Primary isolates of rat AECs (left) or human lung A549 cells (right) were activated with FASL or anti-Fas antibody (FASmAB) as in Fig. 1. Twenty hours later, cell culture medium was collected, freed of detached cells, lyophilized, and analyzed by ELISA specific for ANG II. Values are means of at least 2 separate determinations. * Significantly different from CTL, P < 0.05 (by Student's t-test).



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Fig. 6.   Abrogation of Fas-induced apoptosis of AECs by neutralizing antibodies specific for ANG II. Primary rat AECs (top) or human A549 cells (bottom) were made apoptotic with FASL, FASmAB, or purified ANG II in presence and absence of SAR, caspase inhibitor Z-Val-Ala-Asp-fluoromethylketone (ZVAD-fmk) (19), or neutralizing polyclonal IgG antibodies specific for ANG II (anti-ANG II). Control antibodies were nonspecific isotype-matched IgGs (N.S.IgG) applied at the same concentration as anti-ANG II. Values are means ± SE; n = 3 or more cultures in each of 2 separate experiments. ** Significantly different from CTL, P < 0.01 (by ANOVA and Dunnett's test).


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The ACE inhibitor captopril abrogated Fas-induced apoptosis in A549 cells at doses of the drug readily attained in vivo (20). Because captopril is a sulfhydryl-containing compound, the inhibition of apoptosis was speculated to occur through thiol-mediated blockade of cysteine protease (caspase) activities required for AEC apoptosis (20). However, a subsequent study (23) showed that purified ANG II is capable of inducing ANG II receptor-dependent apoptosis in both A549 cells and primary AECs isolated from rats. More importantly, the experiments reported herein show that the enzymatic activity of ACE and the ability of ANG II to interact with its receptor are not only involved in the signaling of Fas-induced apoptosis but are also required for its execution.

The notion that ANG II binding to its receptor is a required step in the signaling pathway activated by Fas is supported by the ability of the nonselective receptor antagonist (saralasin) and neutralizing anti-ANG II antibodies to completely abrogate (93 and 101% inhibition, respectively) AEC apoptosis in response to Fas (Figs. 1 and 6). Although the inhibitory actions of lisinopril and captopril on AEC apoptosis were slightly less complete (75-85% inhibition; Fig. 1) than that of saralasin, these data do not lessen the importance of ANG II function because ACE, the preferred target of these inhibitors (1), is only one of several dipeptidyl carboxypeptidases capable of cleaving ANG I to ANG II (6, 10). Although the large degree of inhibition exerted by these compounds suggests that ACE may be the primary ANG I cleavage activity expressed by these cells, other enzymes such as chymase or cathepsins (which are not sensitive to ACE inhibitors) might also be present and thus would preclude complete blockage of ANG II formation by these drugs.

The large increase in ANGEN mRNA in response to Fas activation (48-fold for A549 cells and 8-fold for rat AECs; Fig. 2) is consistent with a proposed mechanism in which Fas induces the transcription of ANGEN. Regardless of whether the increased ANGEN mRNA is due to transcriptional activation or changes in transcript stability, the finding that ANGEN antisense oligonucleotides can inhibit apoptosis in response to Fas (Fig. 3) suggests that the level of functional ANGEN mRNA is critical to the process by which Fas signals AEC apoptosis. The fact that the antisense inhibition of apoptosis was not complete (70 and 87% for A549 and primary AECs, respectively; Fig. 3) might be explained by the fact that the transfection efficiency was at best 75% (see METHODS), and thus a fraction of the cells undergoing apoptosis may not have been subject to antisense inhibition.

Regardless, the ANGEN promoter is known to contain an acute-phase response element and to be rapidly responsive to cytokines such as tumor necrosis factor-alpha (3). To our knowledge, however, this is the first report of the induction of ANGEN expression specifically by Fas. In cardiac myocytes, apoptosis induced by mechanical strain ("stretch") was recently shown to be mediated by a mechanism in which increased levels of p53 protein interact directly with the ANGEN promoter to induce its transcription (9, 16); the newly synthesized ANGEN protein is then proteolytically cleaved to ANG II, which induces cardiomyocyte apoptosis through binding to its receptor in a manner entirely analogous to that shown earlier for AECs (23). In some cell types, apoptosis in response to Fas is believed to involve the induction of p53 expression (13), which raises the interesting possibility that Fas activation in AECs might also produce elevated levels of p53 protein. The possibility that the Fas-induced increase in the abundance of ANGEN mRNA (Fig. 2) is due to p53-mediated transcriptional activation is currently under investigation.

In summary, Fas-induced apoptosis of A549 cells or primary cultures of rat AECs was significantly inhibited by nonthiol ACE inhibitors and was completely abrogated by an ANG II receptor antagonist. Activation of Fas significantly increased the abundance of ANGEN mRNA, and transfection of antisense oligonucleotides against ANGEN significantly inhibited Fas-induced apoptosis. Activation of Fas increased the amount of free ANG II in the extracellular space, and neutralizing antibodies specific for ANG II completely blocked the induction of apoptosis by Fas. Together, these data support the hypothesis that Fas-induced apoptosis by AECs requires the induction of ANGEN expression, its proteolytic processing, and subsequent binding of ANG II to at least one of its receptors. They also suggest that therapeutic manipulation of Fas-induced apoptosis in vivo will be feasible with a variety of well-characterized pharmacological antagonists of the renin-ANG system.


    ACKNOWLEDGEMENTS

We thank Drs. P. Denes and D. Vidisagar for support of this project.


    FOOTNOTES

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 Cardiovascular Institute and the Research and Education Foundation, Michael Reese Hospital (Chicago, IL).

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

Address for reprint requests and other correspondence: B. D. Uhal, The Cardiovascular Institute, Michael Reese Hospital, 2929 S. Ellis Ave., Rm. 405KND, Chicago, IL 60612 (E-mail: bdu1{at}earthlink.net).

Received 29 July 1999; accepted in final form 8 September 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Akasu, M., U. Hidenori, A. Kinoshita, M. Sasguri, M. Ideishi, and K. Arakawa. Differences in tissue angiotensin II-forming pathways by species and organs in vitro. Hypertension 32: 514-520, 1998[Abstract/Free Full Text].

2.   Bardales, R. H., S. S. Xie, R. F. Schaefer, and S. Hsu. Apoptosis is a major pathway responsible for the resolution of type II pneumocytes in acute lung injury. Am. J. Pathol. 149: 845-852, 1996[Abstract].

3.   Brasier, A., L. Junyi, and A. Copland. Transcription factors modulating angiotensinogen gene expression in hepatocytes. Kidney Int. 46: 1564-1566, 1994[Medline].

4.   Hagimoto, N., K. Kuwano, H. Miyazaki, R. Kunitake, M. Fujita, M. Kawasaki, Y. Kanika, and N. Hara. Induction of apoptosis and pulmonary fibrosis in mice in response to ligation of FAS antigen. Am. J. Respir. Cell Mol. Biol. 17: 272-278, 1997[Abstract/Free Full Text].

5.   Hagimoto, N., K. Kuwano, Y. Nomoto, R. Kunitake, and N. Hara. Apoptosis and expression of FAS/FAS ligand mRNA in bleomycin-induced pulmonary fibrosis in mice. Am. J. Respir. Cell Mol. Biol. 16: 91-101, 1997[Abstract].

6.   Katwa, L. C., A. Ratajska, J. Cleutjens, G. Zhou, S. Lee, and K. T. Weber. Angiotensin converting enzyme and kininase II-like activities in cultured valvular interstitial cells of the rat heart. Cardiovasc. Res. 29: 57-64, 1995[Medline].

7.   Kuwano, K., R. Kunitake, M. Kawasaki, Y. Nomoto, N. Hagimoto, Y. Nakanishi, and N. Hara. p21 and p53 expression in association with DNA strand breaks in idiopathic pulmonary fibrosis. Am. J. Respir. Crit. Care Med. 154: 477-483, 1996[Abstract].

8.   Lai, K., J. Leung, K. Lai, W. To, V. Yeung, and F. Lai. Gene expression of the renin-angiotensin system in human kidney. J. Hypertens. 16: 91-102, 1998[Medline].

9.   Leri, A., P. P. Claudia, Q. Li, X. Wang, K. Reiss, S. Wang, A. Malhotra, J. Kajstura, and P. Anversa. Stretch-mediated release of angiotensin II induces myocyte apoptosis by activating p53 that enhances the local renin-angiotensin system and decreases the Bc1-2-to-Bax protein ratio In the cell. J. Clin. Invest. 101: 1326-1342, 1998[Abstract/Free Full Text].

10.   Liao, Y., and A. Husain. The chymase-angiotensin system in humans: biochemistry, molecular biology and potential role in cardiovascular diseases. Can. J. Cardiol. 11: 13F-19F, 1995[Medline].

11.   Mason, R. J., and M. C. Williams. Type II alveolar epithelial cells. In: The Lung: Scientific Foundations, edited by R. G. Crystal, and J. B. West. New York: Raven, 1991, p. 235-246.

12.   Molteni, A., W. Ward, C. Ts'ao, N. Solliday, and M. Dunne. Monocrotaline-induced pulmonary fibrosis in rats: amelioration by captopril and penicillamine. Proc. Soc. Exp. Biol. Med. 180: 112-120, 1985[Abstract].

13.   O'Conner, L., and A. Strasser. Fas, p53 and apoptosis. Science 284: 1431-1433, 1999.

14.   Panoskaltsis-Mortari, A., and R. P. Bucy. In situ hybridization with digoxigenin RNA probes: fact and artifacts. Biotechniques 18: 300-307, 1995[Medline].

15.   Phillips, M. I., D. Wielbo, and R. Gyurko. Antisense inhibition of hypertension: a new strategy for renin-angiotensin candidate genes. Kidney Int. 46: 1554-1556, 1994[Medline].

16.   Pierzchalski, P., K. Reiss, and P. Anversa. p53 induces myocyte apoptosis via activation of the renin-angiotensin system. Exp. Cell Res. 234: 57-65, 1997[Medline].

17.   Ponte, P., S. Y. Ng, J. Engel, P. Gunning, and L. Kedes. Evolutionary conservation is in the untranslated regions of actin mRNAs: DNA sequence of a human beta -actin cDNA. Nucleic Acids Res. 12: 1687-1696, 1984[Abstract].

18.   Uhal, B. D. Cell cycle kinetics in the alveolar epithelium. Am. J. Physiol. 272 (Lung Cell. Mol. Physiol. 16): L1031-L1045, 1997[Abstract/Free Full Text].

19.   Uhal, B. D., K. M. Flowers, and D. E. Rannels. Type II pneumocyte proliferation in vitro: problems and future directions. Am. J. Physiol. Suppl. (Oct.) 261: 110-117, 1991.

20.   Uhal, B. D., C. Gidea, R. Bargout, A. Bifero, O. Ibarra-Sunga, M. Papp, K. Flynn, and G. Filippatos. Captopril inhibits apoptosis in human lung epithelial cells: a potential antifibrotic mechanism. Am. J. Physiol. 275 (Lung Cell. Mol. Physiol. 19): L1013-L1017, 1998[Abstract/Free Full Text].

21.   Uhal, B. D., I. Joshi, A. True, S. Mundle, A. Raza, A. Pardo, and M. Selman. Fibroblasts isolated after fibrotic lung injury induce apoptosis of alveolar epithelial cells in vitro. Am. J. Physiol. 269 (Lung Cell. Mol. Physiol. 13): L819-L828, 1995[Abstract/Free Full Text].

22.   Uhal, B. D., C. Ramos, I. Joshi, A. Bifero, A. Pardo, and M. Selman. Cell size, cell cycle, and alpha -smooth muscle actin expression by primary human lung fibroblasts. Am. J. Physiol. 275 (Lung Cell. Mol. Physiol. 19): L998-L1005, 1998[Abstract/Free Full Text].

23.   Wang, R., A. Zagariya, O. Ibarra-Sunga, C. Gidea, E. Ang, S. Deshmukh, G. Chaudhary, J. Baraboutis, G. Filippatos, and B. D. Uhal. Angiotensin II induces apoptosis in human and rat alveolar epithelial cells. Am. J. Physiol. 276 (Lung Cell. Mol. Physiol. 20): L885-L889, 1999[Abstract/Free Full Text].

24.   Ward, W., A. Molteni, C. Ts'ao, and J. Hinz. Captopril reduces collagen and mast cell accumulation in irradiated rat lung. Int. J. Radiat. Oncol. Biol. Phys. 19: 1405-1409, 1990[Medline].

25.   Ward, P., A. Molteni, C. Ts'ao, Y. Kim, and J. Hinz. Radiation pneumotoxicity in rats: modification by inhibitors of angiotensin converting enzyme. Int. J. Radiat. Oncol. Biol. Phys. 22: 623-625, 1992[Medline].


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