Angiotensin II activates MAPK and stimulates growth of human pulmonary artery smooth muscle via AT1 receptors

Nicholas W. Morrell1, Paul D. Upton1, Sailesh Kotecha2, Alyson Huntley2, Magdi H. Yacoub3, Julia M. Polak4, and John Wharton4

1 Section on Clinical Pharmacology and 4 Department of Histochemistry, Imperial College School of Medicine, Hammersmith Hospital, London W12 0NN; 3 Heart Science Centre, Harefield Hospital, Middlesex UB9 6JH; and 2 Department of Child Health, Leicester Royal Infirmary, University of Leicester, Leicester LE2 7LX, United Kingdom


    ABSTRACT
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INTRODUCTION
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To determine a potential role for the renin-angiotensin system in the growth of human pulmonary artery (PA) smooth muscle, we studied the localization of angiotensin (ANG) II-receptor subtypes by autoradiography in sections of human PA and in cultured PA smooth muscle cells (PASMCs) and examined the growth responses to ANG II in vitro. Specific 125I-labeled [Sar1,Ile8]ANG II binding was demonstrated within the pulmonary arterial media, but binding to cultured cells varied between isolates. Binding in tissues and cells was inhibited by the ANG II type 1 (AT1) receptor antagonist losartan but not by the type 2 (AT2) receptor antagonist PD-123319. Microautoradiographic studies indicated that cultured PASMCs exhibit heterogeneity with regard to ANG II binding sites. Addition of ANG II to serum-deprived PASMCs, exhibiting a relatively high level of 125I-[Sar1,Ile8]ANG II binding, led to a dose-dependent stimulation of DNA synthesis at 24 h and protein synthesis at 48 h. ANG II led to an increase in cell size without an increase in cell number. These effects were inhibited by losartan but not by PD-123319. In addition, ANG II led to rapid activation of mitogen-activated protein kinase (MAPK), and ANG II-stimulated DNA synthesis was inhibited by the specific inhibitor of MAPK PD-98059. We conclude that the AT1 receptor is expressed by human PASMCs in vivo and in vitro and is coupled to activation of MAPK and increased DNA and protein synthesis in vitro. These results are consistent with the hypothesis that ANG II may be involved in human pulmonary vascular remodeling.

mitogen-activated protein kinase; vascular remodeling; pulmonary hypertension; angiotensin II type 1 receptors


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
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DISCUSSION
REFERENCES

A PREVIOUS STUDY IN ANIMALS (36) has suggested that the renin-angiotensin system may contribute to the development of pulmonary hypertension. Indeed, long-term administration of the angiotensin-converting enzyme (ACE) inhibitor captopril to patients with pulmonary hypertension has been shown to reduce pulmonary vascular resistance in some studies (1, 30) but not in another (17). More recently, Morrell et al. (24) and others (37) have demonstrated that administration of captopril or the specific angiotensin (ANG) II type 1 (AT1) receptor antagonist losartan markedly attenuated the hemodynamic and structural changes of hypoxia-induced pulmonary hypertension in the rat. Furthermore, it was found that chronic hypoxia increased the expression of ACE, the enzyme mainly responsible for the conversion of ANG I to ANG II, in the walls of small pulmonary arteries undergoing remodeling (22).

These observations have led to the hypothesis that local production of ANG II by ACE in the pulmonary arterial wall could contribute to smooth muscle hypertrophy and/or hyperplasia seen in remodeled hypertensive pulmonary arteries (22). However, although it is recognized that ANG II can stimulate smooth muscle cell (SMC) growth (3, 10, 25) in addition to its traditional role as a vasoconstrictor, the growth response to ANG II is known to be highly variable depending on the species, the vascular bed, and culture conditions (12). Moreover, the growth response can vary widely between isolates (27). This variation may reflect differences in receptor distribution and receptor coupling to signal transduction cascades (38).

The present study sought to determine whether ANG II could be a factor that contributes to hypertensive remodeling of the pulmonary arterial media in humans. The approach involved isolation of segments of the proximal human pulmonary artery (PA) and determination of the tissue distribution of ANG II-receptor subtypes by in vitro autoradiography. Similar segments were used for isolation of medial PASMCs in culture. Ligand binding studies were used to establish the ANG II-receptor binding characteristics in cultured cells, and the growth responses to ANG II were examined. The results demonstrate that isolates of human PASMCs vary considerably in the relative density of ANG II binding sites. In isolates that possess high-level binding, receptors are predominantly of the AT1 subtype and are coupled to activation of mitogen-activated protein kinase (MAPK) and DNA and protein synthesis. Our findings are consistent with the hypothesis that the renin-angiotensin system may contribute to PASMC hypertrophy in human pulmonary hypertension and suggest that inhibition of ANG II-generating pathways may prove useful in the treatment of this condition.


    METHODS
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METHODS
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Reagents and antibodies. Medium 199 (M199), type II collagenase, fetal bovine serum (FBS), antibiotic-antimycotic solution, trypsin-EDTA, and four- and eight-well slide chambers were purchased from GIBCO BRL (Paisley, UK). Optimal cutting temperature mounting medium was obtained from H&E Histological Equipment (Nottingham, UK). Losartan was provided by Merck Research Laboratories (West Point, PA). PD-123319 was provided by Parke-Davis (Wilmington, DE). PD-98059 was purchased from Calbiochem-Novabiochem (Nottingham, UK). 125I-labeled [Sar1,Ile8]ANG II (2,200 Ci/mmol) was purchased from DuPont-NEN (Stevenage, UK); [methyl-3H]thymidine (6.7Ci/mmol) was from ICN Biomedicals (Thame, UK); L-[4-3H]phenylalanine, Hyperfilm 3H autoradiography film, and Hypercoat LM-1 emulsion were from Amersham; ANG II and [Sar1,Ile8]ANG II, monoclonal antibodies to alpha -smooth muscle actin (clone 1A4) and smooth muscle myosin (clone hSM-V), FITC-conjugated anti-mouse IgG, L-phenylalanine, and all other reagents were purchased from Sigma (Poole, UK).

Tissue preparation. Proximal segments of the human PA (main, right and left, or lobar arteries) were obtained from resected lung specimens from patients undergoing lung or heart-lung transplantation for primary pulmonary hypertension (n = 3 men and 1 woman; mean age 29 yr), congenital heart disease (n = 3 men and 2 women; mean age 32 yr), or underlying lung disease (n = 7 men and 2 women; mean age 47 yr) comprising emphysema (n = 4), sarcoid (n = 2), histiocytosis (n = 1), fibrosing alveolitis (n = 1), and cystic fibrosis (n = 1). Additional samples of the proximal PA were obtained from unused donors for transplantation (n = 7 men and 4 women; mean age 31 yr). Further specimens were obtained from patients undergoing lobectomy or pneumonectomy for bronchial carcinoma (n = 4 men; mean age 67 yr). Samples of the proximal PA (pulmonary trunk or right or left PA) were placed in M199 at 4°C for transport from the operating room to the laboratory. An intact section of the PA (~0.5 cm long) was removed from the original specimen in some cases for frozen sections and in vitro autoradiographic studies. The segments were blotted dry and placed in optimal cutting temperature mounting medium on cork boards, then immediately frozen in isopentane cooled by liquid nitrogen. Once frozen, the specimens were stored at -40°C before being sectioned.

The remaining samples of the PA were used for isolation of PASMCs (see Isolation of PASMCs).

In vitro autoradiography of ANG II binding sites. ANG-receptor expression in proximal PA segments (n = 17) was characterized by receptor autoradiography. The diagnoses in this group were as follows: primary pulmonary hypertension (n = 4), congenital heart disease (n = 4), donors for heart-lung transplantation (n = 5), and underlying lung disease (n = 4). Ten-micrometer-thick cryostat sections were incubated in 10 mM sodium phosphate buffer (pH 7.4) containing the labeled antagonist 125I-[Sar1,Ile8]ANG II as previously described (15). As in previous studies on human tissues (15, 16), binding reached an apparent equilibrium within 90 min at 20-22°C (data not shown). Nonspecific binding was defined as that remaining in adjacent sections coincubated with either 10-6 M unlabeled [Sar1,Ile8]ANG II or ANG II. Binding sites were characterized further by incubating consecutive sections with 2.5 × 10-10 M 125I-[Sar1,Ile8]ANG II in the presence of the AT1-receptor antagonist losartan or the AT2-receptor antagonist PD-123319. Macroautoradiographic images were obtained by apposing labeled sections to Hyperfilm 3H for 3-4 days at 4°C.

Further anatomic resolution of the binding sites was achieved by tissue microautoradiography as previously described (16). Sections were incubated with ligand as described above, then apposed to emulsion-coated coverslips for 2-3 wk at 4°C.

Isolation of PASMCs. The arteries were opened to expose the endothelial surface, which was removed by gentle scraping with a scalpel. The surrounding adventitia was then carefully dissected from the tunica media. Cells were derived either from explants or after tissue digestion. For the explant technique, the pulmonary arterial media was cut into ~2-mm cubes and plated onto 25-cm2 tissue culture flasks. Explants were left to adhere overnight and then were maintained in M199, 20% FBS, and antibiotic-antimycotic solution (100 U/ml of penicillin, 100 µg/ml of streptomycin, and 250 ng/ml of amphotericin B). The cells were passed after ~2 wk into a single 75-cm2 flask and grown to confluence in M199-10% FBS.

For tissue digests, the medial layer was cut into small pieces with a scalpel and incubated in type II collagenase (1,000 U/ml) in serum-free medium at 37°C for 4 h. The action of collagenase was stopped by the addition of M199-20% FBS. The cells were then passed through a filter (pore size 100 µm) and centrifuged at 200 g for 5 min, then resuspended in M199-20% FBS before being plated in 25-cm2 flasks. Subsequent passages were carried out at confluence, dividing one flask into four. Cells were used for experiments between passages 3 and 10.

Additional experiments were conducted with a commercially available human PASMC line (Clonetics, TCS Biologicals, Bucks, UK) previously reported (11) to undergo hypertrophy in response to ANG II stimulation. Cells were grown and passaged according to instructions from and with reagents provided by the supplier.

The phenotype of isolated cells was investigated with antibodies to smooth muscle-specific antigens: monoclonal anti-alpha -smooth muscle actin (clone 1A4) and anti-smooth muscle myosin (clone hSM-V). For immunostaining, the cells were grown to subconfluence in eight-well slide chambers. The cells were fixed in acetone at -20°C for 10 min, then washed in phosphate-buffered saline (PBS) for 3 × 5 min. The cells were incubated with primary antibody for 1 h at room temperature, then with anti-mouse FITC-conjugated secondary antibody for 1 h, again at room temperature. Between steps, the slides were thoroughly rinsed in PBS for 3 × 5 min at room temperature. The cells were then mounted in a solution of PBS and glycerol (1:1) and visualized by fluorescence microscopy.

Ligand binding studies on cultured cells. For ligand binding studies in cultured cells, PASMCs were seeded at a density of 20,000 cells/well in 24-well plates. The cells were allowed to grow to 70-80% confluence in M199-10% FBS. For the binding assays, the cells were intially washed twice for 5 min in serum-free M199. The cells were then incubated in M199-0.1% (wt/vol) BSA containing 10-10 M 125I-[Sar1,Ile8]ANG II. Preliminary studies showed that binding reached an apparent equilibrium within 90 min at 37°C (data not shown). Nonspecific binding was defined as that remaining bound to cells incubated with 10-6 M unlabeled [Sar1,Ile8]ANG II, and ANG-receptor subtypes were characterized by incubation with either 10-6 M losartan or 10-6 M PD-123319. The cells were washed twice for 1 min with serum-free M199 at 4°C and lysed with 0.2 M NaOH-0.1% SDS. The lysates were transferred to 5-ml polystyrene vials and counted in a scintillation counter. Protein concentration was assayed with the Bio-Rad DC protein assay (Bio-Rad, North Yorkshire, UK) according to the manufacturer's instructions.

Competition curves were derived by incubating the cells with 10-10 M 125I-[Sar1,Ile8]ANG II in M199-0.1% BSA in the presence of increasing concentrations (10-11 to 10-7 M) of unlabeled [Sar1,Ile8]ANG II. To obtain estimates of maximal binding capacity and dissociation constant, saturation curves were generated by incubating the cells with increasing concentrations of 125I-[Sar1,Ile8]ANG II (10-11 to 10-9 M) in the presence and absence of 10-6 M [Sar1,Ile8]ANG II.

Microautoradiography in cell monolayers. In some isolates, binding of 125I-[Sar1,Ile8]ANG II to single cells was examined by microautoradiography in SMC monolayers. Cells were plated at a density of 2,000/well in four-well slide chambers and grown for 48 h in M199-10% FBS. Monolayers were washed 2 × 5 min in PBS before the addition of M199-0.1% BSA containing 10-10 M 125I-[Sar1,Ile8]ANG II with and without the addition of 10-6 M losartan, 10-6 M PD-123319, or 10-6 M ANG II. After 90 min, the cells were washed 2 × 1 min in M199 and 1 × 10 s in distilled H2O. The slides were then air-dried, and emulsion was applied to the slide with a wire loop. The slides were developed after 7 and 14 days, mounted, and examined by dark- and light-field microscopy.

Growth responses to ANG II. Growth of human PASMCs was determined by [methyl-3H]thymidine and L-[4-3H]phenylalanine incorporation, representing DNA and protein synthesis, respectively. Cells from passages 3-10 were used in all experiments. For [methyl-3H]thymidine studies, the cells were washed with PBS, trypsinized with 0.25% trypsin-EDTA for 5 min, and suspended in M199-10% FBS. The cells were then seeded in 24-well plates at a density of 20,000/well and grown to 70-80% confluence. At this stage, the cells were washed three times with warm PBS and quiesced by incubation with M199-0.1% FBS for 48 h. The medium was then exchanged for fresh M199-0.1% FBS containing 10-10 to 10-6 M ANG II and 0.5 µCi/well of [methyl-3H]thymidine for 24 h with and without the addition of 10-6 M losartan or 10-6 M PD-123319. Additional experiments were performed with ANG II with and without the addition of the selective inhibitor of MAPK activation PD-98059 (3-30 µM) (5). The cells were rapidly washed three times with PBS at 4°C followed by the addition of 1 ml/well of 10% TCA at 4°C for 30 min. The TCA was then discarded, and 0.5 ml of 0.2 M NaOH was added to each well. The plate was stored at 4°C overnight, the resulting lysates were transferred to scintillation vials, and thymidine incorporation determined by scintillation counting.

Protein synthesis was determined at 24, 48, and 72 h with a similar protocol as for [methyl-3H]thymidine incorporation except that the cells were incubated with 0.4 mM L-phenylalanine for 30 min before the addition of 2 µCi/well of L-[4-3H]phenylalanine for the final 24 h of exposure to the test conditions.

For studies of cell proliferation, the cells were incubated with 10-6 M ANG II for 1-4 days. Because ANG II may be degraded by peptidases in culture, fresh ANG II was added daily to achieve a final concentration of 10-6 M. The cells were counted with a hemocytometer, and viability was assessed by trypan blue exclusion.

To determine whether ANG II increased cell size, we measured the relative size distribution by flow cytometry. Cells were grown to 70-80% subconfluence in 75-cm2 flasks and quiesced in 0.1% serum for 3 days before incubation with 10-6 M ANG II for 48 h. The cells were then harvested by trypsinization and centrifugation. Single-cell suspensions of equal density were prepared in serum-free M199 and then immediately measured for relative size distribution based on forward scatter (FACS Vantage, Becton Dickinson). Forward scatter has been shown to correlate with cell size (14).

MAPK activity. Activation of MAPK was assayed by immunoblotting. The cells were grown to 70-80% confluence in 35-mm petri dishes, washed, and then quiesced in M199-0.1% FBS for 48 h. The medium was replaced with fresh M199-0.1% FBS for 90 min and then treated with M199-0.1% FBS either with or without the addition of 10-7 M ANG II. At specified time points (1, 5, 10, 20, 30, 60, and 120 min), the medium was removed and the cells were snap-frozen in liquid nitrogen, then harvested in 0.5 ml of lysis buffer (containing 20 µM E64, 1 µM pepstatin A, 2 µg/ml of aprotinin, 10 µg/ml of leupeptin, 62.5 mM beta -glycerophosphate, 50 mM NaF, 2.5 mM Na3VO4, 0.1% Triton X-100 in 20 mM Tris, and 2 mM EDTA, pH 7.4) with a cell scraper. The lysate was divided into aliquots and stored frozen at -20°C before blotting.

In additional experiments, the effect of PD-98059 on ANG II-stimulated MAPK activation was examined. The cells were treated as above except that 10-7 M ANG II was added for 10 min with and without preincubation (30 min) with 30 µM PD-98059.

For immunoblotting, the samples were thawed and sonicated for 10 min at 4°C to shear DNA and reduce sample viscosity. The samples were then microcentrifuged for 10 min at 13,000 rpm, and the supernatant was collected. Protein content was determined as above. The samples (20 µg of total protein) were subjected to SDS-polyacrylamide gel electrophoresis (12%). The proteins were then electrotransferred to nitrocellulose membrane and incubated in 10% Marvel in Tris-buffered saline-Tween (TBS-T), pH 7.4, overnight at 4°C. The membrane was washed 4 × 10 min in TBS-T, followed by a 1-h incubation with polyclonal anti-phosphorylated MAPK primary antibody (1:1,000; New England Biolabs). These antibodies detect p42mapk and p44mapk only when catalytically activated by phosphorylation at Tyr204. The membrane was again washed before a 1-h incubation with a horseradish peroxidase-conjugated secondary antibody (1:2,000; New England Biolabs). After four further washes in TBS-T, the membrane was incubated in electrochemiluminence reagent (Amersham) for 1 min before being exposed to film for ~2 min.

Statistics. Differences between control and experimental conditions were analyzed by one-way analysis of variance and a post hoc Tukey's honestly significant difference test. Conventional significance was considered to have been achieved when P was <0.05. All results are expressed as means ± SE. Binding curves were analyzed with GraphPad Prism version 2.01 (GraphPad Software, San Diego, CA).


    RESULTS
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Distribution of ANG II-receptor subtypes by autoradiography. To determine the ANG II-receptor subtype(s) in the intact pulmonary arterial media, autoradiographic studies were employed. These studies demonstrated specific binding of 125I-[Sar1,Ile8]ANG II to the pulmonary arterial media in all arteries studied (n = 17; Fig. 1, A-C). This binding was inhibited by incubation with an excess of unlabeled ANG II or with the specific AT1-receptor antagonist losartan. However, binding was unaffected by incubation with 10-6 M PD-123319, the specific AT2-receptor antagonist. These findings indicate that the majority of ANG II binding sites in the normal or diseased human pulmonary arterial media are of the AT1 subtype. Microautoradiographic studies on sections of PA demonstrated an apparently homogeneous pattern of radioligand binding within the media (data not shown).


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Fig. 1.   Autoradiographs of a section of main pulmonary artery from a patient with primary pulmonary hypertension (A-C) and a pulmonary artery smooth muscle cell (PASMC) monolayer (D-F) showing distribution of 125I-[Sar1,Ile8]ANG II binding either alone (total binding; A and D) or in presence of ANG II type 1 (AT1) receptor antagonist losartan (B and E) or AT2-receptor antagonist PD-123319 (C and F). Virtually all activity bound to arterial media (A-C, open arrows), and cell monolayers could be displaced by losartan, whereas PD-123319 had no effect, indicating predominance of AT1-receptor subtype. Relatively little binding was detected in neointima (*) of hypertensive artery. Microautoradiography in cell monolayers demonstrated a mixed population of cells with different binding densities within the same isolate (D-F); intense labeling with 125I-[Sar1,Ile8]ANG II is observed in some cells, whereas neighboring groups of cells had only low-level or no binding (solid arrows). Bars, 3 mm in A-C and 75 µm in D-F.

To assess the possibility that the different PASMC isolates were heterogeneous with respect to ANG II-receptor expression (see ANG II binding sites on cultured human PASMCs), we conducted microautoradiographic studies in cell monolayers in isolates with high and low levels of binding. These studies showed a mixed population of PASMCs within the same isolate, with individual cells exhibiting different binding densities. Relatively more cells demonstrated a higher binding density in those isolates with a high degree of specific binding (Fig. 1, D-F).

ANG II binding sites on cultured human PASMCs. Cells derived by the explant or digest technique yielded cells with the typical morphology of vascular SMCs. The smooth muscle phenotype of these cells was confirmed by positive immunofluorescence with anti-smooth muscle myosin and anti-alpha -smooth muscle actin.

To determine the presence and subtype of ANG II binding sites on cultured cells, we performed radioligand binding studies with 125I-[Sar1,Ile8]ANG II. In five isolates, we detected a high level (>90%) of specific 125I-[Sar1,Ile8]ANG II binding: two from patients with congenital heart disease, one from a patient transplanted for sarcoidosis, and two from control subjects. The relative binding density in these isolates was 81 ± 23 fmol/mg protein (range 41-158 fmol/mg protein). Ligand binding to these isolates could be inhibited by prior incubation with losartan but not with PD-123319 (Fig. 2), suggesting that in vitro, the predominant ANG II receptor was of the AT1 subtype, similar to that in the intact media. Competition binding experiments demonstrated that the binding was both saturable and specific. Analysis of the saturation-binding curves yielded a mean maximal binding capacity of 1,131 ± 72 fmol/mg and a dissociation constant of 0.32 ± 0.05 nM for 125I-[Sar1,Ile8]ANG II (Fig. 3). The relative binding density in these isolates was found to be unaffected by the increasing passage number of cells in culture (repeated measurements made up to passage 13).


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Fig. 2.   Binding density of 125I-[Sar1,Ile8]ANG II to human PASMCs. Cells were incubated with 10-10 M 125I-[Sar1,Ile8]ANG II either alone (total binding) or in presence of an excess of unlabeled [Sar1,Ile8]ANG II (nonspecific binding), AT1-receptor antagonist losartan, or AT2-receptor antagonist PD-123319. Values are means ± SE of 4 wells. Results are representative of 3 separate binding experiments in isolates with relatively high-level binding. * P < 0.001 compared with total binding.



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Fig. 3.   Saturation-binding curve showing specific () and nonspecific (black-triangle) binding of 125I-[Sar1,Ile8]ANG II to human PASMCs grown in 24-well plates. Each point is mean ± SE of 3 wells.

Of the remaining isolates, 21 showed relatively low-level (<50% specific) binding of 125I-[Sar1,Ile8]ANG II, with a mean binding density of 0.76 ± 0.16 fmol/mg protein (range 0.08-2.25 fmol/mg), and 7 demonstrated no specific binding.

Binding studies to the commercial human PA cell line revealed only low-level binding, with a relative binding density of 4.34 ± 0.12 fmol/mg protein.

Growth responses to ANG II and effects of specific receptor antagonists. Incubation with ANG II for 24 h resulted in a dose-dependent increase in [methyl-3H]thymidine in the isolates found to have high-level specific binding sites for 125I-[Sar1,Ile8]ANG II (Fig. 4A). In these cells, the effect of 10-7 M ANG II was significantly inhibited by 10-6 M losartan but was unaffected by the presence of 10-6 M PD-123319, suggesting that the observed increase in DNA synthesis was mediated via the AT1-receptor subtype (Fig. 4B). The addition of ANG II (10-6 M) to these isolates caused no increase in cell number over a 4-day period despite the daily addition of fresh ANG II to the culture medium. Further experiments were conducted for up to 10 days, again with no increase in cell number (data not shown).


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Fig. 4.   ANG II-stimulated [3H]thymidine incorporation in human PASMCs. A: addition of increasing concentrations of ANG II to serum-deprived cells led to a dose-responsive increase in [3H]thymidine incorporation compared with that in control cells maintained in 0.1% fetal bovine serum (FBS). cpm, Counts/min. Values are means ± SE of 4 wells. Results are representative of 3 separate experiments with different isolates. ** P < 0.001 compared with 0.1% FBS. * P < 0.01 compared with 0.1% FBS. B: coincubation of cells with AT1-receptor antagonist losartan (10-6 M) but not with AT2-receptor antagonist PD-123319 (10-6 M) significantly inhibited ANG II (10-9 to 10-7 M)-induced [3H]thymidine incorporation. Values are means ± SE of 4 wells. Results are representative of 3 separate experiments with different isolates. ** P < 0.01 compared with 0.1% FBS. * P < 0.05 compared with 10-7 M ANG II.

In the same isolates, protein synthesis as measured by incorporation of L-[4-3H]phenylalanine was stimulated by ANG II in a dose-dependent manner after 48 and 72 h of incubation (Fig. 5A) but not by 24 h of incubation. The response was again inhibited by losartan but not by PD-123319 (Fig. 5B).


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Fig. 5.   ANG II-stimulated [3H]phenylalanine incorporation in human PASMCs. A: addition of increasing concentrations of ANG II to serum-deprived cells for 48 h led to a dose-responsive increase in [3H]phenylalanine incorporation compared with that in control cells maintained in 0.1% FBS. Values are means ± SE of 4 wells. Results are representative of 3 separate experiments with different isolates. ** P < 0.01 compared with 0.1% FBS. * P < 0.05 compared with 0.1% FBS. B: coincubation of cells with AT1-receptor antagonist losartan (10-6 M) but not with AT2-receptor antagonist PD-123319 (10-6 M) significantly inhibited ANG II (10-7 M)-induced [3H]phenylalanine incorporation at 48 h. Values are means ± SE of 4 wells. Results are representative of 3 separate experiments with different isolates. * P < 0.05 compared with 0.1% FBS.

The above experiments suggested that ANG II induced hypertrophy rather than proliferation of PASMCs. To support this, we measured the relative cell size by flow cytometry in isolates with a high-level binding of 125I-[Sar1,Ile8]ANG II. Incubation with ANG II led to an ~10% increase in mean relative cell size compared with that in control cells (Fig. 6).


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Fig. 6.   Flow cytometry analysis of PASMC size. Incubation with 10-6 M ANG II (solid line) led to a rightward shift in distribution compared with that in control cells (dashed line), indicating an increase in relative size.

In the remaining isolates that demonstrated only low-level or absent binding to 125I-[Sar1,Ile8]ANG II, no measurable increase in [methyl-3H]thymidine or L-[4-3H]phenylalanine incorporation was detected in response to incubation with ANG II.

Role of MAPK. To determine whether MAPK is involved in the ANG II-induced growth response in cells with relatively high-level 125I-[Sar1,Ile8]ANG II binding, we studied the time course of activation of MAPK by immunoblotting and the effect of inhibition of MAPK activation on ANG II-mediated DNA synthesis. Incubation of PASMCs with 3-30 µM PD-98059 led to a dose-responsive inhibition of ANG II-induced [methyl-3H]thymidine incorporation (Fig. 7). Immunoblotting for phosphorylated p42mapk and p44mapk demonstrated rapid (1-min) activation of both isoforms, which peaked at 5 min and declined over the subsequent 10-60 min (Fig. 8A). Pretreatment of the cells with PD-98059 markedly attenuated the ANG II-induced activation of MAPK (Fig. 8B), confirming the inhibitory effect of this compound on MAPK activation.


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Fig. 7.   Role of mitogen-activated protein kinase (MAPK) in ANG II-induced DNA synthesis. ANG II-stimulated [3H]thymidine incorporation was dose dependently inhibited by specific inhibitor of MAPK activation, PD-98059 (PD). * P < 0.01 compared with 10-7 M ANG II.




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Fig. 8.   A: immunoblotting for phosphorylated (phospho) p42 and p44 isoforms of MAPK demonstrated early activation of both isoforms. B: further experiments confirmed that PD-98059 inhibited ANG II-stimulated MAPK activation. A, control; B, 10-min exposure to ANG II (10-7 M); C, 10-min exposure to ANG II after 30-min preincubation with 30 µM PD-98059. Results are representative of 3 separate experiments with different isolates.


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The main findings of this study are that the AT1-receptor subtype is the predominant ANG receptor in the human pulmonary arterial media and isolated human PASMCs and that ANG II is capable of activating MAPK and stimulating DNA and protein synthesis via the AT1 receptor in these cells. We used in vitro autoradiography to show that the human pulmonary arterial media and PASMC monolayers possess specific binding sites for the radiolabeled ANG II analog 125I-[Sar1,Ile8]ANG II. The displacement of this ligand by the specific AT1-receptor subtype antagonist losartan but not by the AT2-receptor antagonist PD-123319 suggests that the human pulmonary arterial media and isolated PASMCs express ANG II receptors predominantly of the AT1 subtype. Furthermore, we found that primary cultures of human PASMCs exhibited heterogeneity in the relative binding density for 125I-[Sar1,Ile8]ANG II between isolates. This heterogeneity may be due to the presence of a mixed population of cells within a given isolate containing varying proportions of cells with relatively high- and low-level binding as demonstrated by microautoradiograpy in cell monolayers.

The distribution of ANG II-receptor subtypes in the human main PA has not been previously studied. Although at least four ANG II-receptor subtypes have been identified, only two, the AT1 and AT2 subtypes, have been studied in detail in vascular tissue. The mRNA for the AT1 subtype has been localized to fetal rat PASMCs (23) and is expressed at lower levels in adult PAs. The AT2 subtype predominates over the AT1 subtype during development of the rat aorta but is expressed at very low levels in adult arteries (33). However, reexpression of the AT2 subtype has been reported in balloon-injured adult rat arteries (13). Furthermore, the AT2 receptor has been localized to rat endothelial cells where it may mediate growth (29). Our autoradiographic studies in tissue sections, receptor binding studies in cultured cells, and microautoradiographic studies in cell monolayers indicate that ANG II binding sites in the media of large human PAs and isolated PASMCs are of the AT1 subtype. This is consistent with a previous report from this laboratory (35) demonstrating predominantly AT1 binding sites in the media of human coronary arteries.

One explanation for the finding of PASMC isolates with high- and low-level 125I-[Sar1,Ile8]ANG II binding is cellular heterogeneity in the expression of ANG II receptors. The concept of cellular heterogeneity within the arterial media is well recognized in the bovine PA (7, 8). The use of microautoradiography in cell monolayers allowed us to demonstrate the presence of individual PASMCs with high- and low-level binding within the same isolate. Isolates with relatively high-level 125I-[Sar1,Ile8]ANG II binding were enriched by a relatively greater proportion of individual PASMCs with a high level of binding. In contrast, in vitro macro- and microautoradiography of radioligand binding in the intact PA did not suggest regional heterogeneity of receptor expression. However, autoradiography does not allow sufficient resolution to confirm differences in ANG II-receptor binding between individual cells in the arterial media. Therefore, possible explanations for our findings include 1) heterogeneity of PASMC AT1-receptor expression in vivo and in vitro and 2) homogeneous PASMC AT1 expression in vivo, with heterogeneity in the capacity of the cells to retain receptor expression in culture. Our data would suggest the latter but cannot exclude the former.

When PASMCs with a relatively high level of 125I-[Sar1,Ile8]ANG II binding were incubated with ANG II, a dose-dependent increase in DNA synthesis was observed at 24 h and in protein synthesis at 48 and 72 h. Stimulation of DNA and protein synthesis by ANG II was inhibited by coincubation with losartan but not with PD-123319, suggesting that ANG II-induced DNA and protein synthesis is mediated via the AT1-receptor subtype. However, ANG II did not increase PASMC number despite prolonged incubation with 10-6 M ANG II and daily addition of fresh ANG II to the culture medium. These findings suggest that stimulation by ANG II leads to hypertrophy rather than hyperplasia in human PASMCs, similar to previous findings in rat systemic vascular SMCs (9, 10). A hypertrophic effect of ANG II in human PASMCs is supported by the increase in relative cell size we observed by flow cytometry.

In further studies, we addressed the potential role of activation of MAPK, an essential part of the pathways that mediate cell growth, in ANG II-stimulated DNA synthesis. These studies demonstrated that a specific inhibitor of MAPK activation, PD-98059, dose dependently inhibited ANG II-induced DNA synthesis. Furthermore, with immunoblotting for phosphorylated p42mapk and p44mapk, we found that ANG II rapidly activated these isoforms, an effect that was inhibited by PD-98059. The time course of activation of MAPK by ANG II is similar to that previously reported in rat aortic SMCs (6). Taken together, these results provide strong evidence for a growth-promoting effect of ANG II on human PASMCs.

A growth effect of ANG II on systemic vascular SMCs is well recognized (3, 9, 18). However, the reported effects of ANG II as a growth factor appear to be highly variable depending on the species (21) and vascular bed from which the cells are derived as well as on the conditions under which the cells are cultured (12). Relatively few studies (3, 20, 21) have been conducted in primary human vascular SMCs. Generally, ANG II has been found to exert relatively weak effects on DNA synthesis in human systemic SMCs (21), some reports (3, 20) suggesting that ANG II requires the presence of serum to produce a mitogenic effect, whereas another (2) reports no effect of the peptide on mitogenesis. None of these previous studies determined the relative density of SMC binding sites for ANG II. Because we observed considerable variation in ANG II binding between isolates, with a minority of isolates demonstrating high-level binding, it is possible that the reported differences in the growth-promoting properties of ANG II in human cells are related to differences in ANG II-receptor density.

In cultured rat SMCs derived from the systemic circulation, DNA synthesis induced by ANG II frequently has a delayed onset (34), suggesting that it exerts its effects mainly via induction of other growth factors (10, 31, 34). Indeed, there is evidence that the hypertrophic or proliferative response to ANG II is determined by the autocrine production of transforming growth factor-beta 1 (10). In the present study, we observed that ANG II stimulated relatively rapid (within 24 h) synthesis of DNA in primary cultures of human PASMCs under serum-deprived conditions. This observation may indicate the presence of diverse signaling mechanisms involved in the growth response to ANG II in rat and human cells, although whether such differences exist remains to be determined.

One previous study (11) reported a weak stimulatory effect of ANG II on protein synthesis as assessed by [3H]leucine incorporation from the same commercial source of human PASMCs used in the present study. In contrast to the previous report, we were unable to demonstrate ANG II-induced DNA or protein synthesis in these cells, although it is unlikely that we were using cells from exactly the same line. The absence of ANG II-stimulated growth in our cell line was probably due to the relatively low binding density.

Studies in animals (22, 24, 32, 36) have suggested a role for the renin-angiotensin system in the development of hypoxia-induced pulmonary hypertension. The ACE inhibitor captopril and the specific ANG II-receptor antagonist losartan prevented the hemodynamic and structural changes of chronic hypoxia-induced pulmonary hypertension in the rat (24, 37). Furthermore, it has been demonstrated that expression of ACE protein and mRNA is increased in small PAs of rats with hypoxia-induced pulmonary hypertension (22), suggesting that increased local production of ANG II may contribute to vascular remodeling in this animal model. In general, inhibition of endogenous ANG II by losartan does not appear to prevent pulmonary hypertension induced by ingestion of the pyrrolizidine alkaloid monocrotaline (4, 32). Interestingly, however, in a recently developed monocrotaline and high-flow model of pulmonary hypertension (26), which is characterized by the formation of a neointima, ACE inhibitors and losartan both delay the neointimal proliferation. Taken together, these studies perhaps indicate a specific role for endogenous ANG II in the complex cellular events leading to pulmonary vascular remodeling.

Evidence for involvement of the renin-angiotensin system in clinical pulmonary hypertension remains limited. Schuster et al. (28) found evidence for increased local expression of ACE in the endothelium and neointima of elastic PAs of patients with primary pulmonary hypertension. A handful of studies (1, 30) in patients with pulmonary hypertension have indicated that inhibition of ACE may reduce pulmonary vascular resistance during long-term administration. The present study has demonstrated that ANG II is capable of stimulating growth of SMCs in at least a subpopulation of cells derived from the human pulmonary circulation and therefore may contribute to pulmonary vascular remodeling in humans. These studies suggest a potential role for the use of AT1-receptor antagonists in the treatment of clinical pulmonary hypertension.


    ACKNOWLEDGEMENTS

This study was supported by British Heart Foundation Grant PG/96121.


    FOOTNOTES

N. Morrell is a Medical Research Council Clinician Scientist Fellow.

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: N. W. Morrell, Section on Clinical Pharmacology, Division of Medicine, Hammersmith Hospital, Du Cane Road, London W12 0NN, UK (E-mail: nmorrell{at}rpms.ac.uk).

Received 10 November 1998; accepted in final form 1 April 1999.


    REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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