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The expression of renin-angiotensin system components and the
elevation of angiotensin-converting enzyme (ACE) in a number of
fibrotic lung diseases suggests angiotensin II (AII) could play a role
in the pathogenesis of pulmonary fibrosis. However, the effect of
AII on lung fibroblasts has not previously been assessed and the
mechanisms by which AII induces cell proliferation in mesenchymal cells are not fully understood. We have examined the ability of
AII to stimulate fetal and adult human lung fibroblast proliferation
in vitro. In particular, we have assessed the receptor subtypes involved and the possible autocrine role of transforming growth factor 
(TGF-) and platelet-derived growth factor (PDGF), two recognized fibroblast mitogens. Angiotensin type 1 (AT1), but not
type 2, receptors were identified on fetal and adult human lung fibroblasts by immunocytochemistry. AII (1 µM) increased DNA synthesis (determined by [3H]thymidine incorporation) in fetal and
adult cells by 211 ± 18% and 150 ± 14%, respectively (p < 0.01),
and was inhibited by a specific AT1 receptor antagonist, Losartan
(74 ± 14%). A proliferative response to AII was confirmed by direct cell counts. Subsequently, fibroblasts were incubated with
neutralizing antibodies to TGF- and PDGF. Anti-TGF- antibodies
inhibited AII-induced DNA synthesis by 73 ± 13%. However, no effect was seen with anti-PDGF antibodies. In conclusion, we have
shown that angiotensin II induces human lung fibroblast proliferation in vitro via activation of the AT1 receptor and involves the autocrine action of TGF-.
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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The vasoactive octapeptide, angiotensin II (AII), has a well-described role in the control of systemic blood pressure and volume homeostasis. There is also evidence to suggest an important role for AII in the fibrotic response to tissue injury. This includes the ability of AII receptor antagonists and angiotensin-converting enzyme (ACE) inhibitors to attenuate cardiac (1) and renal (2) fibrosis in a number of animal models (3). Such effects are often observed in the absence of changes in circulating renin-angiotensin system (RAS) components or blood pressure and are believed to result from an inhibition of local, tissue-based RAS in which AII production is independent of circulating precursors (4).
AII is generated from the proteolytic cleavage of its precursor, angiotensin I by ACE. ACE is expressed predominantly in a membrane-bound form by the vascular endothelial cells of the pulmonary circulation but is also present in plasma. An elevation in bronchoalveolar lavage fluid and/or serum ACE concentrations has been observed in a number of potentially fibrotic lung diseases, including sarcoidosis (5), idiopathic pulmonary fibrosis (6), asbestosis (7), silicosis (7), and the acute respiratory distress syndrome (8, 9). The functional significance of increased ACE is uncertain but could potentially result in increased AII generation within the lung. Indeed, high AII concentrations have been demonstrated in normal rat lung (10) and also increase during radiation-induced pulmonary fibrosis (11). The existence of a local pulmonary RAS is also supported by the expression of angiotensinogen and AII receptors in rat and human lung tissue (12).
AII may regulate tissue fibrosis via the activation of mesenchymal cells. For example, AII stimulates the proliferation of cardiac fibroblasts (15) in vitro via activation of the type 1 AII receptor (AT1). The presence of AT1 receptors has also been demonstrated on cardiac fibroblasts in vitro (16). Most of the profibrotic effects of AII appear to be mediated via this receptor; however, increased AT2 expression on cardiac fibroblasts has been detected in hypertrophied human heart (17, 18), and the balance between the expression of these two subtypes may be critical in determining the response to AII.
AII is rapidly metabolized to two smaller peptides, a heptapeptide, des-Asp1-AII (AIII) and a hexapeptide des-Asp1-Arg2-AII (AIV), which may also have biologic activity. AIV has been shown to induce microvascular endothelial cell proliferation via a specific type IV receptor (19) and AIII upregulated fibronectin mRNA expression in renal interstitial fibroblasts in vitro (20). Their effects on fibroblasts are not known.
One potential mechanism of action for AII is the induction
of other fibroblast growth factors. In vascular smooth cells
(VSMC), the cellular actions of AII have been linked to the
autocrine synthesis of a number of growth factors, including
transforming growth factor beta (TGF-) (21) and platelet-
derived growth factor (PDGF) (22). However, the growth response in this cell type appears to involve a specific balance
between hypertrophic and hyperplastic influences and may
utilize signaling pathways and autocrine mediators distinct
from other mesenchymal cell types. AII has also been shown
to induce TGF- production in cardiac fibroblasts (23), but its
role in mediating the specific effects of AII in these cells is unclear. TGF- has been implicated as a key mediator of pulmonary fibrosis both in animal models and in human disease (24,
25). It is a potent stimulator of procollagen synthesis by lung
fibroblasts in vitro (26) but induces a biphasic mitogenic response, being stimulatory at low concentrations and inhibitory
at higher concentrations (27).
The potential role of angiotensin peptides as growth factors for lung fibroblasts has not previously been studied. To test the hypothesis that AII is mitogenic for these cells and could thus contribute to fibroproliferation in the lung, we have examined the effect of angiotensin II and its metabolites on human lung fibroblast proliferation in vitro. We have also used subtype-specific receptor antagonists and neutralizing antibodies to growth factors to further elucidate the mechanism of AII-induced fibroblast growth.
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METHODS |
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Cell Culture
Human fetal lung fibroblasts. Human fetal lung fibroblasts (HFL-1) were obtained from the American Type Culture Collection (Rockville, MD). HFL-1 cells were subcultured in Dulbeccos's modified eagle medium (DMEM) supplemented with penicillin (200 U/ml) and streptomycin (200 U/ml), and containing 10% newborn calf serum (NCS). Subconfluent cells at passages 15 to 21 were detached using a trypsin (0.05% wt/vol)/EDTA (0.02% wt/vol) solution, centrifuged (300 × g for 7 min at 4° C), and the cell pellet resuspended in DMEM. For DNA synthesis and proliferation experiments, fibroblasts were then seeded onto 96-well plates at a density of 5 × 103/well.
Adult primary lung fibroblasts. Primary human fibroblasts derived from a patient with histologically proven idiopathic pulmonary fibrosis were used for receptor immunocytochemistry and key cell proliferation assays. Briefly, explanted lung tissue was cut into 1-mm3 pieces and placed into 10-cm petri dishes containing DMEM supplemented as for HFL-1 cells with the addition of fungizone. Fresh medium was added to cultures every 3 d during subculture. The fibroblast cultures were characterized immunohistochemically. Staining was negative for epithelial, mesothelial, endothelial, and smooth muscle cell markers. Greater than 95% of cells stained positively for vimentin, and a proportion of these cells also stained positively for alpha-smooth muscle actin, confirming the fibroblast/myofibroblast phenotype of these cells. Sufficient fibroblasts were present within approximately 3 wk and were subsequently passaged as for HFL-1 cells.
Immunocytochemistry
Angiotensin II type 1 (AT1) and type 2 (AT2) receptors were identified on HFL-1 and adult primary fibroblasts by indirect immunofluorescence. Subconfluent cells were plated onto 8-well chamber slides (Nunc; Life Technologies, Paisley, Scotland, UK) in DMEM containing 10% NCS. Cells were allowed to attach for 24 h and then quiesced in serum-free medium for a further 24 h prior to fixation and staining. Cells were washed twice in PBS and fixed in 4% paraformaldehyde for 5 min. After three washes with PBS, the slides were incubated with normal rabbit serum (5%) diluted in phosphate-buffered saline (PBS) for 30 minutes. Slides were then incubated with a polyclonal antibody to either the human AT1 or AT2 receptor (Santa Cruz Biotechnology, Santa Cruz, CA) diluted 1:100 in 1% bovine serum albumin (BSA) in PBS. Rabbit immunoglobulin (IgG) at an equal concentration (Sigma Chemical Company Ltd., Poole, Dorset, UK) served as control antibody. After three washes with PBS, the slides were incubated with fluorescein isothiocyanate (FITC)-conjugated goat antirabbit IgG (Sigma, UK) at a 1:40 dilution in PBS/1% BSA. All antibody incubations were conducted in a humidified chamber at room temperature.
[3H]Thymidine Incorporation Assay
The effect of AII on DNA synthesis was assessed by the incorporation of [3H]thymidine. The optimal timing and length of the [3H]thymidine labeling was established in preliminary experiments. Subsequently, fibroblasts seeded onto 96-well plates were exposed to either
[Sar1]AII, angiotensin III (AIII) or angiotensin (AIV) at a range of concentrations from 10
5 to 1010 M for 20 h in 100 µl DMEM/well.
0.5 µCi [3H]thymidine was added for the final 4 h of exposure in 10 µl
DMEM. Cells were then lysed with 10 µl 0.1 M NaOH solution and
frozen at 
40° C. DNA was harvested onto glass-fiber filters using a 96-well plate cell harvester (Skatron) Filters representing individual wells were inserted into 6-ml scintillation vials and 5 ml of scintillant
(EcoscintA; National Diagnostics, Somerville, NJ), added. To determine the incorporated radioactivity, vials were counted for 2 min using a Minaxi Tri-Carb 4380 series counter (Packard Instruments,
Canberra Packard, Pangbourne, UK). [Sar1][Thre8]AII, a potent AII
antagonist as well as specific nonpeptide antagonists to both AT1
(Losartan; courtesy of Merck, Sharp and Dohme, Hoddeson, UK)
and AT2 (PD123319; Parke-Davis, Morris Plains, NJ), were used to
determine the receptor type responsible for any change in DNA synthesis. Cells were preincubated for 30 min with antagonist (10 µM)
before the addition of 106 M [Sar1]AII. In additional experiments,
DNA synthesis was also assessed in response to AII in the presence or
absence of panspecific neutralizing antibodies to either TGF- isoforms 1 to 3 (30 µg/ml final concentration; R&D Systems, Oxon, UK),
or PDGF AA, AB, and BB isoforms (10 µg/ml; R&D Systems). The
concentrations of antibody were chosen with reference to the manufacturers data as those required to inhibit 80% of TGF- (1 ng/ml) or
PDGF AB (10 ng/ml) activity. Subsequently, the efficacy of each antibody was confirmed in our culture conditions by their ability to inhibit
TGF- or PDGF AB-induced mitogenesis at a range of concentrations.
Assessment of Cell Number
Briefly, subconfluent cells were brought into suspension and seeded
onto 96-well plates, as described above. Cells were incubated with either [Sar1]AII, AIII, or AT-IV at a range of concentrations from 105
to 1012 M for 24, 48, and 72 h. After stimulation the medium was removed and the cells resuspended in 100 µl trypsin/EDTA at room
temperature and counted using a Neubauer hemocytometer (BDH/
Merck). Six wells were counted for each condition.
Statistical Analysis
Results are expressed as mean ± standard error of the mean. For comparison of test and control values, Student's t test was employed. For multiple comparisons, results were assessed by one-way analysis of variance (ANOVA) and significance determined using a Bonferroni correction; p < 0.05 was considered to be significant. Each experiment was repeated on at least three separate occasions with six replications.
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RESULTS |
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Human Lung Fibroblasts Express the AT1 Receptor
The presence of AT1 receptors on human fetal and adult fibroblast cultures was demonstrated by indirect immunocytochemistry (Figures 1a and 1b). Subconfluent HFL-1 cells, cultured in the absence of serum, demonstrated abundant AT1 expression, visible on the cell surface and as intracellular staining. Less marked expression was observed in the adult fibroblasts though the pattern and distribution of staining was similar. No staining was observed in cells incubated with nonspecific IgG antibody and no staining was seen with an antibody directed against the AT2 receptor in either cell type (Figures 1c and 1d). AT2 expression is consistently observed in a mouse 3T3 cell line known to solely express the AT2 receptor (28).
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AII Is Mitogenic for Human Lung Fibroblasts
[3H]Thymidine incorporation was used to assess the effect of
angiotensin peptides on DNA synthesis by fetal and adult human lung fibroblasts cultured in the absence of serum. AII
stimulated DNA synthesis in a dose-dependent manner with a
maximal response seen at 106 M in both fetal and adult cells
(211 ± 5% and 339 ± 6%, respectively, p < 0.01) (Figure 2).
The consistency of the response to AII in fetal cells was confirmed in seven separate experiments with a combined mean
stimulation of 180 ± 64% above control. The angiotensin metabolites AIII and AIV, at a range of concentrations (1010 to
105 M), had no significant effect on DNA synthesis in either
adult or fetal cells, confirming the specificity of the response
to AII (data not shown).
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0.05; **p It can be seen in Figure 3 that an increase in cell number was confirmed by direct cell counting at 24, 48, and 72 h incubation time. A significant increase was observed at all time points after exposure to AII (control cell number was 11.6 × 103, 12.1 × 103, and 12.7 × 103 at 24, 48, and 72 h, respectively), consistent with the increases in DNA synthesis. For subsequent blocking experiments, [3H]thymidine incorporation was assessed in HFL-1 cells because of the increased sensitivity of this assay to detect changes in the mitogenic response.
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Mitogenic Activity of AII Is Mediated via the AT1 Receptor
Losartan (10 µM), a specific AT1 receptor antagonist, significantly inhibited AII-induced DNA synthesis in HFL-1 cells by 74 ± 14% (p < 0.01) (Figure 4). Losartan at concentrations of 10, 1, 0.1, and 0.01 µM inhibited proliferation by 74, 62, 25, and 2%, respectively. The IC50 for this response was 0.84 µM (29). This confirmed that the increase in proliferation was a AT1-receptor-mediated event. [Sar1][Thre8]AII (10 µM), a nonspecific peptide antagonist, completely inhibited the effect of AII (p < 0.01). No inhibition was observed in the presence of the AT2-specific inhibitor PD123319. Losartan and PD123177 alone had no effect on DNA synthesis.
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TGF- Blocking Antibodies Inhibit
To examine the potential role of autocrine growth factors in
mediating the mitogenic response to AII, cells were treated
with panspecific neutralizing antibodies to either PDGF or
TGF- (Figure 5) Anti-TGF- significantly inhibited AII-
induced DNA synthesis in HFL1 cells by 76 ± 11% (p < 0.01).
Neutralizing antibodies to PDGF had no effect, but they inhibited 92 ± 21% of the mitogenic response to PDGF AB (10 µg/ml). The antibodies alone had no effect.
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The proliferative response to exogenous TGF-1 (5 to 20 pM) alone was also determined for HFL1 cells under these experimental conditions. TGF-1 at lower doses increased DNA
synthesis, with a maximal response seen at 5 pM (37 ± 7%, p < 0.01) and was completely abolished by anti-TGF- neutralizing antibodies. The increase in DNA synthesis induced by
TGF-1 was consistently lower than that seen in response to
AII. At higher doses, a reduction in DNA synthesis was observed (data not shown).
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DISCUSSION |
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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A number of experimental and clinical observations support a
role for AII in the fibrotic response to lung injury, and we have demonstrated for the first time a mechanism by which
AII might directly activate lung fibroblasts. Our results show
that AII is mitogenic for both adult and fetal lung fibroblasts
via activation of the AT1 receptor and involves the autocrine
action of TGF-.
Mitogenic Response
AII increased DNA synthesis by both adult and fetal human lung fibroblasts. Changes in DNA synthesis are sometimes observed without progression to cell division, possibly because of a hypertrophic response or during DNA repair. For this reason, a true increase in cell number was confirmed by direct cell counting. AII is rapidly metabolized to two smaller peptides, a heptapeptide, des-Asp1-AII (AIII) and a hexapeptide des-Asp1-Arg2-AII (AIV) and a specific type IV receptor has recently described. However, the effects of these peptides on fibroblast activity have not previously been studied. The specificity of the proliferative response to AII was confirmed by the absence of a response to these peptides at equimolar concentrations.
Angiotensin Receptors
The AT1 receptor has been shown to mediate mitogenesis in cardiac fibroblasts and vascular smooth muscle cells (15, 22). Activation of the AT2 receptor has been shown to have opposing effects in fibroblasts, inhibiting cell growth (30, 31) and promoting apoptosis (32), and the balance between the expression of these two receptor types may be crucial in determining the response to AII. The expression of the AT1, but not the AT2, receptor was confirmed on both fetal and adult human lung fibroblasts. It is possible, however, that the relative expression of these receptor types could alter under different culture conditions. The 5-min exposure time to fixative allows permeabilization of the cells and intracellular staining is observed. This may represent AT1 receptor stores or internalized AT1 receptors after activation (33). Internalization of the receptor would imply the presence of AII and suggests the possibility that these cells are capable of the autocrine synthesis of AII, but this requires further study. De novo generation of AII by cardiac fibroblasts has been demonstrated in vitro (34). No staining was seen for the AT2 antibody.
The proliferative response to AII was inhibited by both [Sar1][Thre8]AII, a nonspecific AII antagonist, and Losartan (AT1-specific). Although there was a trend towards a greater inhibition by [Sar1][Thre8]AII, this was not significant. The IC50 for Losartan was comparable to that described for vascular smooth muscle cells (29). The involvement of the AT2 receptor is essentially excluded by the lack of inhibition with PD123319. Taken together, these results confirm that AII- induced mitogenesis is mediated by the AT1 receptor. It is, however, possible that signaling via alternative receptors might contribute to this response.
Role of Transforming Growth Factor Beta
TGF- isoforms are both mitogenic and potent stimulators of
procollagen synthesis in lung fibroblasts in vitro (26, 27). Moreover, an important role in vivo is strongly supported by
the expression of TGF-1-3 at sites of fibrosis in human lung and the ability of TGF- antibodies to attenuate lung fibrosis in animal models (35). TGF-1 is synthesized by cardiac fibroblasts in response to AII (23); however, its role in mediating
the proliferative response to AII has not previously been explored. In vascular smooth muscle cells, TGF- did not contribute to a mitogenic response (36) induced by AT-II but appears to regulate AT-II-induced collagen synthesis (37). Such
studies highlight important differences in the cellular response
to AT-II between cell types.
Panspecific antibodies to TGF-1-3 effectively attenuated the
proliferative response to AII in lung fibroblasts, but TGF-1
alone at a range of concentrations produced a biphasic response
that was considerably less than that produced by AII. These observations are consistent with similar reports by our group in
HFL-1 cells in which all TGF- isoforms were mitogenic at concentrations as great as 40 pg/ml and to a comparable degree (27). We speculate that the stimulation of proliferation by angiotensin II may depend on several autocrine loops, including TGF-. These factors may include cytokines or growth factors as well as extracellular and cell surface adhesion molecules, which are known to participate in mitogenesis. Thus, TGF- alone
may not promote as large a proliferative response because these
other synergistic elements are absent. Further studies are required to elucidate this mechanism. A similar observation has
been made in renal tubular cells, but, similarly, no mechanism
has yet been determined (38). It is interesting to speculate that
AII may be one of a number of growth factors that contributes
to the enhanced TGF- expression at sites of injury.
Possible Relevance to Respiratory Disease
Increases in serum and BALF ACE in fibrotic lung disease
might represent one mechanism by which AII generation
could increase after lung injury. It is possible that these
changes result from the nonspecific shedding of ACE from the
endothelial membrane. However, additional enzymes can
cleave angiotensinogen and angiotensin I to AII, and a recent
report suggests chymase may be a major AII-generating enzyme in the lung (39). Despite this, increased ACE expression
in circulating monocytes and pulmonary macrophages in sarcoidosis (40), and the relationship between ACE and other markers of the fibrotic response in this disorder, strongly support a role for this enzyme. Increases in AII in irradiated rat
lung
which mirror the deposition of collagen in this model
more directly implicate AII (11). Further studies are required
to identify additional cell types capable of generating AII directly or expressing RAS components in the lung, and to confirm the activation of a local RAS after lung injury and the ensuing fibrotic response.
In summary, we have shown that AII is mitogenic for human lung fibroblasts, and this observation supports the hypothesis that AII is a potential profibrotic mediator in the
lung. The mitogenic effect is mediated by the AT1 receptor
expressed in adult and fetal cells and appears to involve the
autocrine action of TGF-. ACE inhibitors and AT1 receptor
antagonists are already licensed for clinical use and could have
a role in the treatment of pulmonary fibrosis.
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Footnotes |
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Correspondence and requests for reprints should be addressed to Dr Richard P. Marshall, Centre for Cardiopulmonary Biochemistry and Respiratory Medicine, Rayne Institute, 5, University Street, London WC1E 6JJ, UK. E-mail: Richard. Marshall{at}ucl.ac.uk
(Received in original form July 1, 1999 and in revised form November 24, 1999).
Acknowledgments: Supported by the Wellcome Trust.
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References |
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REFERENCES
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