Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2003 Dec;163(6):2523-30.
doi: 10.1016/S0002-9440(10)63607-3.

Essential roles for angiotensin receptor AT1a in bleomycin-induced apoptosis and lung fibrosis in mice

Xiaopeng Li  1 , Heather Rayford, Bruce D Uhal
Affiliations

Essential roles for angiotensin receptor AT1a in bleomycin-induced apoptosis and lung fibrosis in mice

Xiaopeng Li et al. Am J Pathol. 2003 Dec.

Abstract

Apoptosis of alveolar epithelial cells (AECs) has been implicated as a key event in the pathogenesis of lung fibrosis. Recent studies demonstrated a role for the synthesis and binding of angiotensin II to receptor AT1 in the induction of AEC apoptosis by bleomycin (BLEO) and other proapoptotic stimuli. On this basis we hypothesized that BLEO-induced apoptosis and lung fibrosis in mice would be inhibited by the AT1 antagonist losartan (LOS) or by targeted deletion of the AT1 gene. Lung fibrosis was induced by intratracheal administration of BLEO (1 U/kg) to wild-type C57BL/6J mice. Co-administration of LOS abrogated BLEO-induced increases in total lung caspase 3 activity detected 6 hours after in vivo administration and reduced by 57% BLEO-induced caspase 3 activity in blood-depleted lung explants exposed to BLEO ex vivo (both P < 0.05). Co-administration of LOS in vivo reduced DNA fragmentation and immunoreactive caspase 3 (active form) in AECs, measured at 14 days after intratracheal BLEO, by 66% and 74%, respectively (both P < 0.05). LOS also inhibited the accumulation of lung hydroxyproline by 45%. The same three measures of apoptosis and lung fibrosis were reduced by 89%, 85%, and 75%, respectively (all P < 0.01), in mice with a targeted disruption of the AT1a receptor gene (C57BL/6J-Agtr1a(tm1Unc)). These data indicate an essential role for angiotensin receptor AT1a in the pathogenesis of BLEO-induced lung fibrosis in mice and suggest that AT1 receptor signaling is required for BLEO-induced apoptosis of AECs in mice as it is in rat and human AECs.

PubMed Disclaimer

Figures

Figure 1.
Figure 1.
Detection of DNA fragmentation, activation of caspase 3, and alveolar type II pneumocytes in mouse lung. Deparaffinized lung sections were prepared from mice instilled intratracheally 14 days earlier with sterile saline (A, C, and E) or BLEO (B, D, F, and G). The sections were subjected to ISEL of fragmented DNA (A and B) or IHC with antibodies against the active form of caspase 3 (E–H) or with the type II cell-specific antibody MNF116 (18, C and D). G: Higher magnification of active caspase 3 labeling in F. H: Active caspase 3 labeling in mice treated with BLEO and LOS, an AT1 receptor antagonist. Note ISEL and active caspase 3 labeling in cells in the corners of alveolar walls in the lungs of BLEO-treated mice (B, F, and G, arrowheads) but not in saline-treated mice (A and E) or in mice treated with BLEO and LOS (H). Note also the co-localization of MNF116 (C) with anti-caspase 3 IHC (G) or ISEL (B) in BLEO-treated lungs. D reveals labeling of the type II cell marker MNF116 in relatively normal regions of BLEO-treated lung (D, right) but not in more severely affected regions (D, left). See text for details. Original magnifications: ×400 (A, B, C, G); ×200 (D); ×100 (E, F, H).
Figure 2.
Figure 2.
Histology of mouse lungs at 14 days after instillation of BLEO. A–C: Hematoxylin and eosin preparations of mouse lung instilled intratracheally 14 days earlier with sterile saline (A), BLEO (B), or BLEO and LOS (C). See text and Materials and Methods section for details. Original magnifications, ×200.
Figure 3.
Figure 3.
The AT1-selective receptor antagonist LOS inhibits BLEO-induced activation of caspase 3. A: BLEO was instilled intratracheally into normal mice with and without LOS in the intratracheal instillate (see Materials and Methods). Six hours later, the lungs were perfused to remove blood, excised, and the enzymatic activity of caspase 3 was measured in lung homogenates. B: Lung explants were prepared from normal mouse lung tissue perfused before excision (see Materials and Methods). BLEO (25 mU/ml) was applied in serum-free culture medium for 24 hours in the presence or absence of LOS (10−6 mol/L). Bars are the means ± SEM of n = 6; *, P < 0.05 versus control (CTL); **, P < 0.05 versus BLEO+LOS by analysis of variance and Student-Newman-Keul’s test.
Figure 4.
Figure 4.
AT1 receptor blockade inhibits DNA fragmentation and caspase 3 activation in lung epithelial cells 14 days after BLEO instillation. Normal mice were given a single intratracheal instillation of BLEO in the presence or absence of LOS in the instillate. LOS also was administered thereafter daily intraperitoneally. Fourteen days later, lung sections were prepared and labeled by ISEL (A) or by IHC for the active form of caspase 3 (B). Labeling was quantitated in cells within the alveolar surfaces (see Figure 1C ▶ ). Bars are the means ± SEM of n = 6; *, P < 0.01 versus control (CTL); **, P < 0.05 versus BLEO by analysis of variance and Student-Newman-Keul’s test.
Figure 5.
Figure 5.
AT1 receptor blockade inhibits lung collagen accumulation at 14 days after BLEO instillation. Normal mice were administered BLEO in the presence or absence of LOS as described in Figure 3 ▶ . Fourteen days later, total lung collagen was determined by assay of total hydroxyproline (HP) in hydrolyzed lung tissue (see Materials and Methods). Bars are the means ± SEM of n = 6; *, P < 0.05 versus control (CTL) by analysis of variance and Student-Newman-Keul’s test.
Figure 6.
Figure 6.
Mice deficient in angiotensin receptor AT1a exhibit reduced DNA fragmentation and caspase 3 activation in lung epithelial cells 14 days after BLEO instillation.. Normal [wild type (w.t.)] or heterozygous AT1a knockout mice (+/−) were administered BLEO intratracheally as in Figure 3 ▶ . Fourteen days later, lung sections were prepared and labeled by ISEL (A) or by IHC for the active form of caspase 3 (B), which were quantitated as described in Figure 4 ▶ and Materials and Methods. Bars are the means ± SEM of n = 5; *, P < 0.001 versus wild-type unchallenged (w.t. − BLEO); **, P < 0.01 versus wild-type challenged (w.t. + BLEO) by analysis of variance and Student-Newman-Keul’s test.
Figure 7.
Figure 7.
Mice deficient in angiotensin receptor AT1a exhibit reduced lung collagen accumulation in response to BLEO instillation. Normal [wild type (w.t.)] or heterozygous AT1a knockout mice (+/−) were administered BLEO intratracheally as in Figure 3 ▶ . Fourteen days later lung tissue was fast-frozen, hydrolyzed, and total collagen was measured by hydroxyproline assay (HP) as described in Materials and Methods. A: HP data are expressed as a percentage of the corresponding control (− BLEO). B: Data are expressed as the absolute amount of HP per left lung. Bars are the means ± SEM of n = 5; *, P < 0.01 versus untreated (− BLEO) by analysis of variance and Student-Newman-Keul’s test.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Selman M, King T, Pardo A: Idiopathic pulmonary fibrosis: prevailing and evolving hypotheses about its pathogenesis and implications for therapy. Ann Intern Med 2001, 134:136-151 - PubMed
    1. Gauldie J, Kolb M, Sime P: A new direction in the pathogenesis of pulmonary fibrosis? Respir Res 2002, 3:1-3 - PMC - PubMed
    1. Haschek WM, Witschi HP: Pulmonary fibrosis: a possible mechanism. Toxicol Appl Pharmacol 1979, 51:475-487 - PubMed
    1. Adamson IYR, Young L, Bowden DH: Relationship of alveolar epithelial injury and repair to the induction of pulmonary fibrosis. Am J Pathol 1988, 130:377-383 - PMC - PubMed
    1. Hagimoto N, Kuwano K, Nomoto Y, Kunitake R, Hara N: Apoptosis and expression of FAS/FAS ligand mRNA in bleomycin-induced pulmonary fibrosis in mice. Am J Respir Cell Mol Biol 1997, 16:91-101 - PubMed

Publication types