INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

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.

	METHODS

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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 10⁵ 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.

	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).

View larger version (105K): [in this window] [in a new window]   Figure 1.   Angiotensin type 1 receptor expression human fetal (a) and adult (c) lung fibroblasts in culture. Indirect double layer immunofluorescence using a polyclonal rabbit antibody to the human AT1 receptor was employed to demonstrate expression in quiescent subconfluent fibroblast monolayers. Rabbit IgG served as control primary antibody to demonstrate nonspecific staining in both fetal (b) and adult (d ) cultures.

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).

View larger version (20K): [in this window] [in a new window]   Figure 2.   Effect of AII on DNA synthesis in lung fibroblasts. Human fetal and adult lung fibroblasts were exposed to AII (1010 to 105 M) for 20 h. [3H]Thymidine (0.5 µCi/well) was added for the final 4 h of the incubation period. Control cells were exposed to medium alone. Values are representative results from one of three independent experiments, each of six determinations, and are shown as mean ± SEM disintegrations per minute (× 103). *p  0.05; **p  0.01.

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.

View larger version (13K): [in this window] [in a new window]   Figure 3.   Fibroblast proliferation in response to AII. Proliferation was assessed in serum-deprived fetal and adult lung fibroblasts exposed to AII (106 M) for 72 h by direct cell counting. Values are expressed as mean ± SEM percentage stimulation above control cells treated with medium alone for the same time. *p  0.05; **p  0.01.

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.

View larger version (14K): [in this window] [in a new window]   Figure 4.   Effect of AII receptor antagonists on AII-stimulated increases in DNA synthesis. [3H]Thymidine incorporation was determined in HFL-1 cells preincubated with either [Sar1][Thre8]AII, a nonspecific AII antagonist, or Losartan/PD123319 (105 M), AT1 and AT2 receptor antagonists, respectively, prior to treatment with AII (106 M). Values are expressed as mean ± SEM percentage stimulation above control cells treated with medium alone. *p  0.05 compared with cells treated with AII alone.

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.

View larger version (14K): [in this window] [in a new window]   Figure 5.   Effect of anti-PDGF or anti-TGF neutralizing antibodies on AII-stimulated increases in DNA synthesis. [3H]Thymidine incorporation was assessed in HFL-1 cells in the presence of pan-specific blocking antibodies to TGF-1-3 (30 µg/ml; top panel ) or PDGF isoforms (10 µg/ml; bottom panel ). Cells were preincubated with antibody for 30 min prior to treatment with AII (106 M). Values are representative results from one experiment and are shown as mean ± SEM percentage stimulation above control cells treated with medium alone. *p  0.05, **p  0.01.

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).

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

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 lungwhich 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.