Differential effects of formoterol on thrombin- and PDGF-induced proliferation of human pulmonary arterial vascular smooth muscle cells

Human PAVSM cells were dissociated from pulmonary arteries from failed donor lungs that were obtained from the National Disease Research Interchange (NDRI) (Philadelphia, PA), in accordance with procedures approved by the University of Pennsylvania Committee on Studies Involving Human Beings as previously described [20, 25]. Briefly, a segment of human pulmonary artery just proximal to the lung entry was removed under aseptic conditions, cleaned from connective and fat tissues and dissected as follows: the media of pulmonary artery was dissected from the adventitia and intima and subjected to an enzymatic digestion in 10 ml of buffer containing 0.2 mM CaCl₂, 640 U/ml collagenase, 1 mg/ml soybean trypsin inhibitor and 10 U/ml elastase for approximately 60 min in a shaking water bath at 37°C. The cell suspension was filtered through 105 μm Nytex mesh, and the filtrate was washed with equal volumes of cold Ham's F-12 medium (Life Technologies, Grand Island, NY) supplemented with 10% FBS (HyClone, Logan, UT). Cells were plated on tissue culture plates covered with Vitrogen (Cohesion Technologies Inc., Palo Alto, CA). Cells were cultured in Ham's F-12 media supplemented with 10% FBS (Becton Dickinson, Bedford, MA), 100 U/ml penicillin, and 0.1 mg/ml streptomycin.

For chronic hypoxia experiments, cells were maintained either in normoxic (21% O₂, 5% CO₂) or in hypoxic (1% O₂, 5% CO₂) conditions for 7 days in complete media and then for 48 h in serum-free media supplemented with 0.1% BSA. 1% O₂ was used as we described [25] to reproduce the tissue oxygen levels of moderate chronic hypoxia [32, 33] based on data obtained from the rat chronic hypoxia model of PH [34, 35]. To avoid re-oxygenation, fresh complete or serum-free media was pre-equilibrated at 1% O₂, before adding to PAVSM cells grown under hypoxia. Primary human PAVSM cells in subculture during the second through tenth cell passages were used. All experiments were performed using a minimum of three different cell cultures. Each human PAVSM cell culture was established using pulmonary arterial tissue from a single human donor.

Cells grown under normoxia or chronic hypoxia were serum deprived for 48 h, treated with 0.1, 1, or 10 ng/ml PDGF-BB, 1 U/ml thrombin, 0.2, 2, or 20 μM racemic, (R,R), or (S,S) formoterol, or diluent for 18 h followed by DNA synthesis analysis using the BrdU incorporation assays as we described [25, 36‐38]. Briefly, cells were incubated with 10 μM BrdU for 24 h, fixed with 3.7% paraformaldehyde (Polysciences, Inc., Warrington, PA) for 15 min and permeabilized with 0.1% Triton X-100 for 30 min at room temperature. Following denaturation of DNA with 4 N HCl (3 min at room temperature), incubated for 1 h at 37^oC with 2 μg/ml murine anti-BrdU primary (Becton Dickinson, San Jose, CA) and 10 μg/ml Texas Red-conjugated anti-mouse secondary (Jackson ImmunoResearch Laboratories, West Grove, PA) antibodies for 1 h at 37°C to detect BrdU-positive cells. To detect the total number of nuclei, cells were incubated with 1 μg/ml DAPI. Then, cells were visualized using Eclipse Nikon TE2000E fluorescent microscope (200x magnification), and automatic counts of BrdU-positive and total number of cells were performed using Image-Pro Plus 5.1 software.

Whole cell lysates were prepared using a buffer comprised of 40 mM HEPES (pH 7.5), 120 mM NaCl, 1 mM EDTA, 10 mM sodium pyrophosphate (Na₄P₂O₇ x 10H₂O), 10 mM β-glycerophosphate, 50 mM NaF, 1.5 mM Na₃VO₄, 1% Triton X-100, EDTA-free inhibitor cocktail. Protein contents were measured using a Bio-Rad protein assay reagent kit. Equal amounts of lysate, adjusted for protein content, were subjected to SDS-PAGE and then immunoblot analysis. The blots were exposed to anti-phospho-S6 ribosomal protein, anti-total S6, anti-phospho Akt Ser-473, anti-total Akt, anti-phospho ERK, anti-total ERK, anti-phospho S6K1 T-389, and anti-total S6K1 antibodies (Cell Signaling Technology, Inc., Beverly, MA). All antibodies were in 20 mM Tris (pH 7.5), 150 mM NaCl (TBS) plus 0.5% Tween 20 (TBST), and all incubations were for overnight at 4°C. After 3 washes in TBST, the nitrocellulose membranes were exposed to horseradish peroxidase-conjugated secondary antibody (Boehringer-Mannheim, Indianapolis, IN), washed five times in TBST and visualized using enhanced chemiluminescence (ECL) (Amersham, Arlington Heights, IL).

Data points from each condition are represented as the mean values ± SE. Three independent repetitions were performed for each experimental condition. Statistical analysis was performed using StatView software. Statistically significant differences among groups were assessed with the analysis of variance (ANOVA) (Bonferroni-Dunn) with values of p < 0.05 sufficient to reject the null hypothesis for all analyses. All experiments were designed with matched control conditions within each experiment to enable statistical comparison as paired samples.

Because formoterol inhibits proliferation of human bronchus fibroblasts and airway smooth muscle cells [9, 14], we examined the effects of formoterol and its enantiomers on the proliferation of human PAVSM cells under serum-depleted conditions and in the presence of thrombin and PDGF, confirmed PAVSM mitogens that are involved in PH pathogenesis [20, 39, 40]. Consistent with our published data [20], serum-deprived PAVSM cells had modest levels of DNA synthesis that were significantly increased by treatment with PDGF and thrombin (Figure 1A). Racemic formoterol inhibited DNA synthesis in serum-deprived human PAVSM cells in a concentration-dependent manner (Figure 1B). (R,R) formoterol had a greater inhibitory effect on human PAVSM cell proliferation compared to racemic formoterol (IC₅₀ ~ 1 μM and ~ 4 μM, respectively). (S,S)-formoterol had modest inhibitory effect (IC₅₀ ~ 20 μM) and attenuated DNA synthesis only when used in maximal dose. (R,R) and, to a lesser extent, racemic formoterol significantly inhibited thrombin-induced human PAVSM cell proliferation (IC₅₀ for (R,R) formoterol ~ 20 μM), and (S,S) formoterol had no significant effect (Figure 1C). Both racemic and (S,S) formoterol did not provide 50% inhibition of cell proliferation even when administrated in high doses (20 μM). Interestingly, racemic formoterol and both formoterol enantiomers had no effect on PDGF-induced proliferation of human PAVSM cells (Figure 1D). These data demonstrate that formoterol inhibits basal and thrombin-induced, but not PDGF-stimulated human PAVSM cell proliferation and suggest that growth inhibitory effects of formoterol depend predominantly on its (R,R), but not (S,S) enantiomer.

Because chronic hypoxia is one of the confirmed triggers of PAVSM cell proliferation and pulmonary vascular remodeling in PH [2, 41], we next examined whether formoterol modulates chronic hypoxia-induced PAVSM cell proliferation. As we demonstrated previously [25], chronic hypoxia increased basal and PDGF-induced human PAVSM cell proliferation compared to normoxia-maintained cells (Figure 2A and B, respectively). Importantly, (R,R)-formoterol inhibited chronic hypoxia-induced PAVSM cell proliferation by the level comparable to normoxia-maintained cells, racemic formoterol had lesser inhibitory effect, and no significant differences in cell proliferation were observed in (S,S) formoterol- vs. diluent-treated PAVSMC under chronic hypoxia (Figure 2A). Interestingly, 10 μM (R,R) and racemic formoterol attenuated PAVSM cell proliferation in the presence of 0.1 ng/ml PDGF, but had little effect on the proliferation induced by higher PDGF doses (1 and 10 ng/ml) either under chronic hypoxia or normoxia (Figure 2B) demonstrating that PDGF-induced PAVSM cell proliferation is not susceptible to formoterol. Taken together, these data demonstrate that formoterol attenuates basal, but not PDGF-induced human PAVSM proliferation under chronic hypoxia and that formoterol acts predominantly through its (R,R) enantiomer.

Since the bronchodilatory functions of formoterol require binding of its (R,R) enantiomer with specific β₂AR [42], we next examined whether binding with β₂AR contributes to anti-proliferative effects of formoterol in human PAVSM cells. Serum-deprived cells were treated with the β₂AR blocker propranolol in the presence of 0.2-20 μM of (R,R), (S,S) and racemic formoterol, or diluent and then subjected to DNA synthesis analysis using the BrdU incorporation assay. As seen in Figure 3 (grey bars), (R,R), racemic, and, to a lesser extent, (S,S)-formoterol inhibited DNA synthesis in human PAVSM cells depending on agent concentration. Importantly, propranolol completely reversed inhibitory effects of racemic, (R,R)-, and (S,S)-formoterol on PAVSM cell proliferation (Figure 3, black bars). These data demonstrate that binding with β₂AR is required for formoterol-dependent inhibition of human PAVSM cell proliferation.

Because ERK1/2 signaling is involved in human PAVSM cell proliferation, we next determined the effects of formoterol and its enantiomers on ERK1/2 activatory phosphorylation. As seen in Figure 4A, (R,R) and, to a lesser extent, racemic, but not (S,S) formoterol inhibited basal ERK1/2 phosphorylation. Because PDGF and thrombin, but not chronic hypoxia, markedly activate ERK1/2 in PAVSM cells [20, 25, 43, 44] (Figure 4B), ERK1/2 activation and since (R,R) formoterol demonstrated maximal inhibitory effects on human PAVSM cell proliferation and basal ERK1/2 phosphorylation compared to racemic and (S,S) formoterol, we next evaluated effects of (R,R) formoterol on ERK1/2 phosphorylation induced by PDGF and thrombin. As seen in Figure 4C, D, (R,R) formoterol abrogated thrombin-induced ERK1/2 phosphorylation while having little effect on ERK1/2 phosphorylation induced by PDGF over a range of concentrations (0.1 - 10 ng/ml). Similarly, both (R,R) and racemic formoterol failed to inhibit PDGF-induced ERK1/2 phosphorylation either under normoxia or chronic hypoxia conditions (data not shown). Taken together, these data demonstrate that racemic and (R,R) formoterol down-regulate basal and thrombin-induced, but not PDGF-dependent ERK1/2 phosphorylation in human PAVSM cells.

Next, we examined whether formoterol modulates mTOR signaling in human PAVSM cells by assessing mTORC1-specific ribosomal protein S6 and mTORC2-specific S473-Akt phosphorylation. Consistent with our published data [20, 25], serum-deprived human PAVSM cells had modest S6 phosphorylation levels that were markedly increased by thrombin and PDGF (Figure 5A). In contrast, PDGF, but not thrombin, promoted S473-Akt phosphorylation (Figure 5A) suggesting that both PDGF and thrombin activate mTORC1 signaling in human PAVSM cells while only PDGF increases mTORC2 activity. As we reported previously [25], exposure to chronic hypoxia led to a marked increase in mTORC1-dependent P-S6K1 and P-S6 and mTORC2-dependent P-S473-Akt (Figure 5B) demonstrating that chronic hypoxia activates both mTORC1 and mTORC2 pathways in human PAVSM cells. Interestingly, racemic formoterol and both (R,R) and (S,S) formoterol enantiomers had no effect on basal, PDGF- and thrombin-induced S6 phosphorylation either under normoxia or chronic hypoxia (Figure 5C). PDGF-induced S473-Akt phosphorylation was also comparable in diluent-, (R,R)-, (S,S)-, and racemic formoterol-treated cells (Figure 5D). Collectively, these data demonstrate that formoterol has little effect on mTORC1 and mTORC2 signaling pathways in human PAVSM cells.