Introduction

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In health, pulmonary vascular tone and remodeling are controlled by the balanced local release of vasoactive mediators. In pulmonary hypertension (PH) these control mechanisms are lost leading to vasoconstriction and proliferation of vascular smooth muscle (VSM). Endothelin (ET)-1, a 21 amino-acid peptide, is formed from big-ET-1 by the action of membrane-bound metalloproteases called "ET-converting enzyme(s)" (ECE) (1). ET-1 is both a potent vasoconstrictor (1) and comitogen/proliferation factor (4, 5) for VSM and has therefore been implicated in the pathogenesis of several, if not all, forms of PH (6). Although the main source of ET-1 is considered to be the endothelial cell (1), many cell types in vitro can release this peptide, including VSM cultured from systemic vessels (7). In the case of VSM, ET-1 release can be stimulated by vasoactive mediators such as angiotensin-II (Ang-II) (7, 8), growth factors such as transforming growth factor (TGF)-, platelet-derived growth factor (PDGF) (7, 8), and inflammatory agents including thrombin (11) and cytokines (10). Where studied, the release of ET-1 by VSM cells appears to be regulated at the level of prepro-ET-1 messenger RNA (mRNA) transcription (7). Importantly, the amounts of ET-1 released by VSM cells under inflammatory conditions are equivalent to those produced by the endothelium (10). Further, inasmuch as the number of VSM cells in a remodeling vessel is potentially greater than the number of endothelial cells, endogenous ET-1 production by these cells becomes increasingly important. Despite the interest in ET-1 and PH, no studies to date have established that human pulmonary VSM cells are able to synthesize this peptide in vitro, although histologic evidence suggests that they may produce ET-1 in disease states associated with PH. In human primary PH (PPH), postmortem studies have demonstrated an upregulation of prepro-ET-1 mRNA not only in endothelial cells but also in the VSM (12). In addition, in rodent models of PH associated with congestive cardiac failure or hypoxia, prepro-ET-1 mRNA was also located to pulmonary VSM (13, 14), suggesting synthesis in these cells.

The aims of this study were therefore twofold. First, we addressed the possibility that human pulmonary artery smooth-muscle (HPASM) cells release ET-1 in a manner that regulates proliferation. The demonstration of such an autocrine role for ET-1 in HPASM cells would be important for understanding the pathogenesis of PH and therefore for future therapy. Second, we investigated the influence of current treatments of PH on ET-1 release from HPASM cells.

	Materials and Methods

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Cell Culture

Specimens of human pulmonary artery from healthy segments of lung were obtained from patients undergoing pulmonary resection at the Royal Brompton Hospital. Under sterile conditions, vessels were dissected clean of adventitia and the endothelium was removed mechanically with a scalpel blade and cut into 3- to 4-mm² sections. Dulbecco's modified Eagle's medium (DMEM) was added to the tissue in a tissue culture flask and supplemented with 15% heat-inactivated fetal calf serum (FCS), L-glutamine (2 mM), streptomycin (100 µg/ml), penicillin (100 U/ml), amphotericin B (2.5 µg/ml), and nonessential amino acids (L-alanine, L-asparagine, L-aspartate, L-glutamate, glycine, L-proline, and L-serine, at the manufacturer's recommended concentrations (Life Technologies, Paisley, UK). Cells were incubated at 37°C in an atmosphere of 5% CO₂ and 95% air, and were confluent after approximately 4 wk. HPASM cells were identified by characteristic "hill and valley" morphology and, for representative cultures, confirmed by staining with fluorescein isothiocyanate-labeled anti-smooth-muscle actin antibody. Because endothelial cells are an important source of ET-1, contamination by these cells was excluded by staining with the endothelial cell marker CD31, followed by flow cytometry and immunocytochemistry. Cells between passages 2 and 9 were seeded onto 96-well plates.

Measurement of ET-1 and Big-ET-1 Release

To assess the effect of FCS on ET-1 release, cells were seeded onto 96-well culture plates, left to grow to full confluence (10,000 cells/ well), serum-deprived for 24 h (DMEM as described earlier without FCS and with 0.1% bovine serum albumin), and then treated with the same medium supplemented with 10% FCS for another 24 h. The supernatant was removed and the concentration of ET-1 or big-ET-1 was measured using commercially available enzyme-linked immunosorbent assays (ELISAs) (R&D Systems, Abingdon, UK; and Biomedica, Vienna, Austria) either immediately or after storage at 80°C.

Effects of Drugs on ET-1 Release from HPASM

To investigate the effects of various drugs on serum-stimulated ET-1 release, HPASM cells were incubated with 10% FCS with or without drugs for 24 h. The medium was analyzed for ET-1 or big- ET-1 release as described earlier. Drugs used were phosphoramidon (50 µM), an ECE inhibitor; cicaprost (1 × 10¹⁰ to 1 × 10⁶ M), a stable synthetic prostacyclin analogue; diltiazem (1 × 10⁷ to 1 × 10⁴ M), a calcium-channel antagonist; sodium nitroprusside (SNP, 1 × 10⁷ to 1 × 10⁴ M), a nitric oxide donor; or adenosine (1 × 10⁷ to 3 × 10⁴ M). All release experiments were carried out in the presence of peptidase inhibitors captopril (1 µM), bestatin (1 µM), thiorphan (1 µM), and bacitracin (3 µg/ml) to prevent degradation of ET-1 by endogenous peptidases (14). Cell viability was assessed in representative experiments by measuring the ability of cells to reduce 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) to formazan, a measure of cell respiration. At the end of the experiment, after supernatant removal, cells were incubated with MTT (1 mg/ml) for 15 min at 37°C. After removal of MTT, the cells were solubilized in 100 µL of dimethyl sulfoxide. Formazan levels were assessed by measurement of optical density (OD) at 550 nm. None of the treatments used affected viability measured in this way.

RNA Extraction and Polymerase Chain Reaction

To investigate whether increased release of ET-1 from HPASM cells was due to upregulation of prepro-ET-1 mRNA or ECE-1, reverse transcription (RT) polymerase chain reaction (PCR) was performed. HPASM cells were cultured in six-well plates for 0.5 to 24 h with or without varying concentrations of FCS. As a positive control, the cytokines tumor necrosis factor (TNF)- and interferon (IFN)- (both at 10 ng/ml) were added for 4 h because this combination has been shown to upregulate prepro-ET-1 mRNA at this time point in systemic VSM cells (10). Total RNA was extracted according to the method of Chomczynski and Sacchi (15) with minor modifications. The quantity of 0.6 µg of total RNA was converted to complementary DNA using reverse transcriptase (Promega, Southampton, UK). PCR was performed using primers selected for prepro-ET-1, ECE-1b/c (primers known to detect the isoforms of this enzyme present in systemic VSM cells [10]), and -actin from sequences published in Genbank (10) using a Crocodile III thermocycler (Appligene Oncor, Chester-le-Street, UK). Annealing temperatures and cycle numbers were 53°C and 25 cycles, 54°C and 28 cycles, and 57.5°C and 19 cycles for prepro-ET-1, ECE-1b/c, and -actin, respectively. Sequences used were as follows: for prepro-ET-1, sense 5'-GATGCCAATGTGCTAGCCAA-3' and antisense 5'-CTGATGGAAGCCAGTGAAGA-3'; for ECE-1b/c, sense 5'-GATGTCGACGTACAAGC-3' and antisense 5'-CTGTTGGAGTTCTTGGAATC-3'; and for -actin, sense 5'-GGCACCACACCTTCTACAATG-3' and antisense 5'-CAGGAAGGAAGGTTGGAAGAG-3'. PCR products were analyzed on 1% agarose gels and bands were analyzed for densitometry. Results are expressed as density (OD) of prepro-ET-1 or ECE-1b/ c against that of -actin in the same samples.

Proliferation Experiments

Methyl-[³H]thymidine uptake. Cells were seeded at 30% confluency (3,000 cells/well) onto 96-well plates and serum-deprived for 72 h to achieve quiescence. The medium was then changed with or without the addition of FCS (0.3 to 20%) and cells were incubated for 24 or 72 h to determine the proliferative response to FCS at these time points. To determine the effects of exogenous ET-1, ET-1 (10¹⁰ to 10⁷ M) was added together with increasing concentrations of FCS for 24 or 72 h. For experiments investigating the role of endogenously released ET-1, the ET-A-specific antagonist BQ123 (10 µM) or the ECE inhibitor phosphoramidon (50 µM) was added 1 h before the addition of 0 or 10% serum. The 10% FCS was chosen because this gave the maximum growth response as well as the greatest release of ET-1 over 72 h. The quantity of 1 µCu of methyl-[³H]thymidine in 20 µl serum-free medium was added to each well for the last 6 h of the incubation. Reactions were terminated by freezing the plates at 80°C. Finally, cells were lysed by thawing and the DNA incorporating labeled thymidine was harvested on glass-fiber filters using a cell harvester (Packard, Meriden, CT). Complete cell lysis was confirmed by microscopy. The incorporation of radioactivity was measured with an automatic -scintillation counter (Packard).

Cell number estimation. Cells were seeded onto 96-well plates at 50% confluency (corresponding to approximately 5,000 cells/well). They were serum-deprived for 72 h as described earlier. Cells were treated with drugs and serum as described earlier for 72 h. Incubations were terminated by removing the medium and washing the cells twice in phosphate-buffered saline, followed by freezing at 80°C. Cell numbers were determined by fluoromimetric analysis of DNA levels using the CYQUANT assay (Molecular Probes, Cambridge, UK) with a standard curve constructed by serial dilutions of a known number of cells, determined using a hemocytometer.

Materials

DMEM, penicillin, streptomycin, glutamine, nonessential amino acids, sodium pyruvate, and FCS were obtained from GIBCO BRL Life Technologies (Paisley, UK). ET-1 was purchased from The Peptide Institute, Inc. (Barnet, UK). Phosphoramidon, diltiazem, bacitracin, thiorphan, captopril, MTT, bestatin, and SNP were obtained from Sigma Chemical Co. (Poole, Dorset, UK). The cytokines TNF- and IFN- and the ET-1 immunoassay ELISA were purchased from R&D Systems. The big-ET-1 ELISA kit was obtained from Biomedica. Cicaprost was a kind gift from Schering AG (Berlin, Germany). Primers for PCR were constructed by GIBCO BRL Life Technologies. BQ123 was purchased from Alexis Biochemicals (Nottingham, UK).

Statistical Analysis

All data are reported as means ± standard error of the mean (SEM) from n experiments. Statistical analysis was made by one-sample t test for normalized data or by one-way analysis of variance (ANOVA) as appropriate, using GraphPad Prism version 2.01 (GraphPad Software, Inc., San Diego. CA). P values were considered significant at < 0.05.

	Results

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Effect of FCS on ET-1 Release by HPASM Cells

Incubation with FCS for 24 or 72 h stimulated HPASM cells to release ET-1 into the supernatant with a maximum response seen at 10% FCS (24 h; 16.4 ± 7.7 pg/ml compared with 3.2 ± 1.5 pg/ml at 0% FCS; n = 5, P < 0.05). Results obtained at 72 h are shown in Figure 1. Phosphoramidon (50 µM) inhibited the release of ET-1 stimulated by 10% FCS with a concomitant increase in big-ET-1 production, confirming de novo synthesis of ET-1 (Figure 2). Importantly, neither big-ET-1 nor ET-1 was detectable in unconditioned medium or in unconditioned medium plus serum (0.3 to 20%; data not shown; n = 3 different sources of FCS). (See Figure 2B for medium plus 10% FCS.)

View larger version (26K): [in this window] [in a new window]   Figure 1.   The effect of FCS on release of ET-1 from HPASM cells. HPASM cells were cultured for 72 h in the presence of increasing concentrations of FCS. ET-1 released into the supernatant was measured by ELISA. Data are means ± SEM from three separate patients' cells, repeated in triplicate. *P < 0.05, **P < 0.01 by one-way ANOVA compared with 0% FCS.

View larger version (16K): [in this window] [in a new window]   View larger version (18K): [in this window] [in a new window]   Figure 2.   The effect of phosphoramidon on release of ET-1 or from HPASM cells treated with or without 10% FCS. HPASM cells were incubated either with or without 10% FCS for 24 h in the presence of phosphoramidon (phos, 50 µM). ET-1 (A) or big-ET-1 (B) levels were measured in the supernatant by ELISA. In some wells cells were absent in order to assess ET-1 or big-ET-1 levels in medium or FCS alone. Data are means ± SEM from two separate patients' cells, repeated in triplicate. In one condition the SEM is too small to be shown clearly.

Effect of FCS on Transcription of Prepro-ET-1 mRNA

FCS (10%) caused an upregulation of prepro-ET-1 mRNA steady-state levels at 1 and 2 h, but not at 4 or 24 h after addition of the stimulus (Figure 3; 24 h not shown). However, addition of cytokines TNF- and IFN- (10 ng/ml each) did increase prepro-ET-1 levels at 4 h. We could not detect any variation in the steady-state levels of ECE-1b/c in cells treated with or without FCS at any of the time points measured (data not shown).

View larger version (30K): [in this window] [in a new window]   View larger version (42K): [in this window] [in a new window]   Figure 3.   (A) RT-PCR analysis of prepro-ET-1 mRNA after stimulation with FCS. A representative gel showing the time course for prepro-ET-1 mRNA after stimulation with 10% FCS: lane 1, control 0% FCS at time 0; lane 2, 0% FCS at 1 h; lane 3, 10% FCS at 1 h; lane 4, 0% FCS at 2 h; lane 5, 10% FS at 2 h. A comparison is made with mRNA for -actin at the same time points. Similar results were found when this experiment was repeated using cells from a different patient. (B) RT-PCR analysis of prepro-ET-1 mRNA after treatment with the cytokines TNF- and IFN-. HPASM cells were cultured with TNF- (10 ng/ml) and IFN- (10 ng/ml) for 4 h in the presence of 10% FCS: lane 1, 10% FCS control; lane 2, 10% FCS plus cytokines. A comparison is made with mRNA for -actin.

Effect of FCS on Proliferation of HPASM Cells

FCS stimulated thymidine uptake in HPASM cells in a concentration-related manner at 24 and 72 h after treatment. At 24 h a threshold response to FCS was seen at 0.3% (not shown) and a maximum effect at 10% (medium without FCS, 1,166 ± 575 counts per min [cpm]; medium plus 10% FCS, 10,184 ± 5,055 cpm; n = 4). Similarly, at 72 h a threshold response was seen at 0.3% and a maximum at 10% (medium without FCS, 350 ± 133 cpm: medium plus 10% FCS, 7,445 ± 5,689 cpm). In our hands, there was a large degree of variation in the levels of thymidine taken up by cells cultured from individual patients (as evidenced by the magnitude of standard errors calculated from data combined from all patients). This variation may also have been due, in part, to different phenotypes of smooth-muscle cell being present in our cultures. However, we were unable to observe such differences, at least under the microscope. Despite this, in each case, cells from individual patients responded in the same way to FCS (Figure 4A). In line with observations made using thymidine uptake as a measure of proliferation, FCS induced increases in HPASM cell numbers at 72 h in a concentration-dependent fashion (Figure 4B; n = 3).

View larger version (19K): [in this window] [in a new window]   View larger version (21K): [in this window] [in a new window]   Figure 4.   (A) The effect of FCS on methyl-[3H]thymidine uptake in HPASM cells. HPASM cells were cultured for 72 h in the presence of increasing concentrations of FCS. The quantity of 1 µCu of methyl-[3H]thymidine was added for the last 6 h of the incubation. Data shown are from one representative patient's cells, repeated in triplicate. In several conditions the SEM is too small to be shown clearly. Cells from three further patients showed a similar response curve (data not shown). (B) The effect of FCS on cell number. HPASM cells were cultured in the presence of increasing concentrations of FCS. Cell number was then determined by CYQUANT assay. Data are means ± SEM from three separate patients' cells, repeated in six wells. *P < 0.05, **P < 0.01 by one sample t test compared with 0% FCS control.

Effect of Exogenous ET-1 on Proliferation of HPASM Cells

In the absence of FCS, ET-1 had no effect on thymidine uptake (at 24 or 72 h) or cell number (at 72 h) in HPASM cells (n = 5; data not shown). However, when cells were coincubated with 0.3% FCS, ET-1 induced concentration-dependent increases in thymidine uptake (effective concentration to give 50% maximum response: 5.6 × 10¹⁰ M) at 72 h, with a maximum response seen at 10⁸ M ET-1 (Figure 5). However, costimulation of cells with ET-1 plus 0.3% FCS for the shorter time of 24 h had no effect on HPASM cell growth (data not shown). Similarly, at 72 h, ET-1 (1 × 10⁷ M) increased cell number by 63 ± 25% (n = 5). At higher concentrations of FCS (3 or 10%), additional pro-proliferative effects of ET-1 were not observed (data not shown; n = 5).

View larger version (12K): [in this window] [in a new window]   Figure 5.   The effect of exogenous ET-1 on methyl-[3H]thymidine uptake by HPASM cells. HPASM cells were cultured for 72 h in the presence of increasing concentrations of ET-1 and 0.3% FCS. The quantity of 1 µCu of methyl-[3H]thymidine was added for the last 6 h of the incubation. Data are means ± SEM from four separate patients' cells, repeated in triplicate. *P < 0.05, **P < 0.01 by one sample t test compared to 0.3% FCS control.

Role of Endogenously Produced ET-1 in FCS-Induced Proliferation of HPASM Cells

BQ123 (10 µM) or phosphoramidon (50 µM) inhibited thymidine uptake by 23.6 ± 5.9% and 17.1 ± 7.4%, respectively, when cells were cultured in the presence of 10% FCS for 72 h (Figure 6A; n = 5). For HPASM cells cultured from one patient out of six, BQ123 paradoxically increased thymidine uptake by 41%. This observation was considered to be atypical and was therefore excluded from the group. There was no effect of either BQ123 or phosphoramidon on basal thymidine uptake (0% FCS). In line with data on thymidine uptake, BQ123 or phosphoramidon inhibited the 10% FCS-induced increase in cell number by 27.5 ± 4.8% and 8.7 ± 1.7%, respectively (Figure 6B). Neither BQ123 nor phosphoramidon had an effect on cell number in the absence of FCS.

View larger version (29K): [in this window] [in a new window]   View larger version (17K): [in this window] [in a new window]   Figure 6.   (A) Inhibition of FCS-induced methyl-[3H]thymidine uptake by HPASM cells in the presence of BQ123 or phosphoramidon. HPASM cells were cultured for 72 h in the presence of 10% FCS, with or without the ET-A receptor antagonist BQ123 (10 µM) or phosphoramidon (50 µM). The quantity of 1 µCu of methyl-[3H]thymidine was added for the last 6 h of the incubation. Results are expressed as percentage control (the difference in methyl-[3H]thymidine uptake [cpm] between cells treated with 10% FCS and 0% FCS alone). Data are means ± SEM from five different patients' cells, repeated in triplicate. *P < 0.05 by one sample t test. (B) Inhibition of FCS-induced increase in cell count by BQ123 or phosphoramidon. HPASM cells were cultured under the same conditions as in A. Cell number was determined by the CYQUANT method. Results are expressed as percentage control (the difference in cell number between cells treated with 10% FCS and 0% FCS alone). Data are means ± SEM from three separate patients' cells, repeated in triplicate. *P < 0.05 by one sample t test.

Effect of Vasodilator Drugs on ET-1 Release by FCS-Stimulated HPASM Cells

The increased production of ET-1 induced by 10% FCS was significantly inhibited by diltiazem (100 µM) and cicaprost (0.1 µM). SNP (up to 100 µM) and adenosine (up to 300 µM) appeared to have no effect (Table 1).

	Discussion

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We have shown that HPASM cells secrete ET-1 in response to FCS and that this is, in part, responsible for FCS-induced proliferation. These data suggest an autocrine role for ET-1 as a regulator of the proliferation of this cell type. Clearly, this may be important in the development of PH, particularly in the development of vascular wall remodeling.

Although an autocrine role for ET-1 has been proposed previously, to our knowledge this is the first experimental evidence substantiating this hypothesis in HPASM cells. Nevertheless, pulmonary artery smooth-muscle cells cultured from rats with idiopathic PH, associated with elevated ET-1 levels, have enhanced growth characteristics that were partially inhibited by incubation with BQ123 (16). Similarly, transfection of rat aortic smooth-muscle cells with an ET-1 expression plasmid resulted in overexpression of ET-1 and increased growth compared with untransfected or vector-alone-transfected cells, an effect that was inhibited by BQ123 (17). Further, in human umbilical vein endothelial cells, addition of anti-ET-1 antibody reduced serum-induced DNA synthesis (18). In addition to these reports showing a role for ET-1 in hyperplasia, endogenous production may stimulate hypertrophy in rat cardiomyocytes (19).

In the current study we have shown that serum increased ET-1 production by HPASM cells. A number of previous studies have shown that serum also stimulates endothelial cells in culture to release ET-1 (20, 21) as well as systemic VSM (9). The identity of the active factor(s) in serum is not known. However, using human umbilical vein endothelial cells, others have shown that serum is more potent than plasma as a stimulus for ET-1 release (21). These observations suggest that factors released from platelets or factors involved with activation of the clotting cascade may be responsible for stimulating ET-1 release. Indeed, such factors, including TGF-₁ (20), PDGF (21), Ang-II (7), or thrombin (22), have been shown to increase ET-1 release from cultured cells. Further, platelets themselves stimulate endothelial cells to release ET-1 (23).

In the current study, de novo production of ET-1 was confirmed by inhibition with phosphoramidon (with a concomitant increase in the precursor big-ET-1). In addition, we found that as well as increasing ET-1 synthesis, serum elevated the mRNA level at 1 and 2 h after stimulation. Interestingly, this increase in mRNA was not apparent at 4 or 24 h after stimulation. This may be explained by the predicted instability of prepro-ET-1 mRNA (24), which has a half-life of approximately 15 min (1). It may be that the transient increase in mRNA for ET-1 cannot fully explain the continuous increase in peptide released over time. Thus, other mechanisms of synthesis and release may be acting to prolong ET-1 production. However, there was no increase detected in mRNA levels for ECE-1b/c in HPASM cells stimulated with serum, ruling out increased levels of ECE underlying the increase in ET-1 production. Nevertheless, it should be noted that serum could affect levels of other ECE enzymes, such as ECE-II (3), in our cells. In support of our findings, a similar transient increase in prepro-ET-1 mRNA was observed in human omental VSM cells treated with Ang-II (7) or human endothelial cells treated with thrombin (25). By contrast, other stimulants of ET-1 release, including TGF- (26) or TNF- and IFN- (10), appear to increase prepro-ET-1 for more prolonged periods of time. In addition to transcriptional regulation, post-transcriptional control mechanisms of ET-1 production have been described. Hu and colleagues reported that high-density lipoproteins (HDL) stimulated the production and secretion of ET-1 from bovine aortic endothelial cells by a post-transcriptional mechanism involving activation of phosphokinase C (27). Because HDL is present in serum, a similar mechanism may be acting to stimulate ET-1 production in HPASM cells stimulated with serum. Finally, there is evidence, at least in endothelial cells, that vesicles store and release ET-1 under certain stimuli (28). There are, as far as we are aware, no reports of similar mechanisms in smooth-muscle cells, although Barnes and Turner have recently reported the striking colocalization of ECE-1 and -actin in human smooth-muscle cells, similar to the appearance in endothelial cells, suggesting that vesicular release may indeed be possible (29).

ET-1 stimulates HPASM cells to proliferate (30) via activation of ET-A receptors. We found that ET-1 required low levels of serum to be present to induce proliferation. This comitogenic property of ET-1 is well recognized (31). It appears that ET-1 is unable to initiate cells to enter the S phase of the cell cycle directly, but acts as a progression factor (5). Others have reported that the conditions of growth-arrest of cultured cells are important in determining the growth-promoting effect of ET-1 (32). At high serum levels (more than 3%) we found that the promitogenic effect of ET-1 was lost, possibly due to the overriding stimulus of serum.

In addition to the pro-proliferative effects of exogenous ET-1 on HPASM cells we found that endogenous production of ET-1 contributed to the action of serum in these cells. In fact, we found that both BQ123 and phosphoramidon inhibited proliferation induced by serum. However, the inhibitory effects of BQ123 were more pronounced than those of phoshoramidon. BQ123 is a potent and specific inhibitor of ET-A receptors, whereas phosphoramidon is a relatively weak inhibitor of ECE that also inhibits the degradation of ET-1, which may account for its reduced effectiveness compared with BQ123 (33). In addition, there may also be phosphoramidon-insensitive isoforms of ECE present in HPASM cells (9).

We did not look at the effect of an ET-B receptor antagonist on proliferation of HPASM cells. Although there is evidence for a role of the ET-B receptor in mediating contraction of human pulmonary artery vessels in vitro (34), there are only a few, contradictory, reports suggesting a role for this receptor subtype in proliferation of human VSM cells, and none in HPASM. In addition, we were unable to demonstrate the presence of ET-B receptor mRNA in HPASM cells by RT-PCR under the conditions used in these experiments (unpublished observation).

The pro-proliferative effects of endogenously released ET-1 in HPASM cells demonstrated here may have direct relevance to our understanding of PH in humans. Calcium-channel blockers and prostacyclin have been shown to decrease mortality in certain subgroups of patients with PPH (35, 36). We found that a calcium-channel blocker (diltiazem) and a prostacyclin-mimetic (cicaprost) greatly reduced ET-1 release by HPASM cells. Interestingly, Langleben and colleagues have recently published clinical data that may support our in vitro findings (37). In their study, long-term administration of intravenous prostacyclin in patients with PPH resulted in a more favorable pulmonary arterial/venous ratio of ET-1 measured in plasma. This would suggest that prostacyclin either increased clearance or decreased production of ET-1 in the diseased lungs. Further, the clinical benefit of prostacyclin in treatment of PH is greater than can be explained by its vasodilator properties and may extend to reduction in smooth-muscle cell hyperplasia (36). The importance of ET-1 in this process has been suggested by animal studies in which ET-1 receptor antagonists have not only reduced PH but also resulted in reversal of vascular remodeling and right ventricular hypertrophy (38). The clinical use of ET-1 receptor antagonists confirms the importance of this mediator in human forms of PH (39). Our findings suggest that such compounds would be of benefit not only in the pulmonary vasodilation observed in these studies but also in modulating remodeling. Our results also provide a rationale for at least part of the beneficial actions of prostacyclin and possibly also calcium-channel blockers in the treatment of PPH.

In summary, we have shown that HPASM cells release endogenous ET-1 in response to serum, which we believe to have pathophysiologic relevance. This endogenously released ET-1 is, at least in part, responsible for the proliferation induced by serum. We have thus identified an autocrine role for ET-1 in HPASM. Because ET-1 appears to be a crucial mediator in the development of PH/vascular wall remodelling, these findings have important implications for understanding the pathogenesis of PH and therefore for future management.