PH is a progressive disease with poor prognosis, the pathological manifestations of which include vasoconstriction and pulmonary vascular remodeling [5, 45]. PH may be familial, idiopathic, or associated with other diseases. Notably, patients with COPD-associated PH have higher morbidity and reduced survival compared to other COPD patients [3, 4]. Pulmonary vascular remodeling in PH is associated with marked medial thickening of small muscular PAs due, at least in part, to increased proliferation of PAVSM cells. PAVSM cell proliferation in COPD-associated PH is caused by multiple factors including persistent hypoxia and increased production of growth factors and pro-inflammatory cytokines [2, 31, 46]. Our study demonstrates that β2AR agonist formoterol inhibits proliferation of PAVSM cells induced by thrombin and chronic hypoxia, but not PDGF. Anti-proliferative activity of formoterol requires binding with β2AR, and (R,R) enantiomer of formoterol shows improved anti-growth effects compared to racemic and (S,S) formoterol. We also report that formoterol inhibits basal and thrombin-induced activation of ERK1/2, but has no effect on mTOR signaling in human PAVSM cells.
Deregulated PAVSM cell proliferation is one of the major pathological components of pulmonary vascular remodeling in PH. The long-term β2AR agonist formoterol, which is currently in use as bronchodilator in COPD, shows anti-proliferative activities in human airway smooth muscle cells and bronchus fibroblasts [9, 14]. In the present study, we explored effects of formoterol on human PAVSM cell proliferation caused by different stimuli involved in PH pathogenesis [2, 31, 47] and found that the growth-inhibitory potency of formoterol highly depends on extracellular stimuli. Thus, formoterol and, especially, its (R,R) enantiomer, inhibited proliferation of non-stimulated PAVSM cells under chronic hypoxia, decreased thrombin-induced proliferation, but had no significant effect on PDGF-dependent PAVSM cell growth.
The mechanisms of β2AR-dependent regulation of cell proliferation are relatively unexplored and appear to be highly cell type-specific. β2AR-induced PKA activation, while inhibiting proliferation in the majority of cell types including vascular smooth muscle cells [8–11, 48, 49] stimulates proliferation of human uveal melanoma cells  and cardiomyocyte hypertrophy . Activation of β2AR-cAMP signaling also up-regulates Epac1, the predominant Epac isoform in VSM cells . Epac1 synergizes with PKA in inhibiting VSM cell proliferation , but increases DNA synthesis in macrophages and prostate cancer cells [52, 53]. Our data show that formoterol-dependent inhibition of human PAVSM cells requires its binding with β2AR. Accordingly, (R,R) formoterol, which has much higher receptor affinity and greater potency to induce β2AR-Gs-dependent cAMP production and PKA activation compared to (S,S) formoterol [8, 54], demonstrates greater anti-proliferative effects than formoterol racemate while (S,S) formoterol has modest effects on PAVSM cell proliferation.
Emerging evidence shows that, in addition to classical Gs-cAMP pathway, β2AR may interact with Gi proteins that, in contrast to Gs, leads to reduction of cAMP levels and inhibition of PKA-dependent signaling . Currently, no evidence exists about involvement of Gi in PAVSM cell proliferation and pulmonary vascular remodeling in PH. In contrast, Gs-dependent activation of cAMP-PKA signaling is well documented in human ASM and PAVSM cells upon formoterol treatment and is required for bronchodilatory and vasodilatory effects of formoterol on COPD patients and for inhibition of SM cell proliferation [7–11, 56]. Gi overexpression, however, has been reported in heart and aorta of spontaneously hypertensive rats; and Gi suppression with pertussis toxin attenuated development of high blood pressure in this model [55, 57–59] suggesting differential mechanisms of β2AR signaling in heart vs. pulmonary vasculature.
The signaling pathways underlying formoterol-dependent inhibition of cell proliferation are not well evaluated. We and others previously demonstrated that thrombin promotes human PAVSM cell proliferation via ERK1/2 signaling while PDGF acts via activation of two major pro-proliferative pathways, PI3K-mTOR and MEK-ERK1/2 . In the majority of cells, including VSM, cAMP-dependent activation of PKA and Epac inhibits ERK1/2 signaling via modulating activities of small GTPases Raf-1 and Rap-1 downstream of Ras [15, 61, 62] clearly demonstrating functional cross-talk between ERK1/2 and β2AR-cAMP cascades.
Interestingly, we found that formoterol markedly inhibits thrombin-, but not PDGF-induced ERK1/2 phosphorylation. A possible explanation is that PDGF, in addition to ERK1/2, also promotes strong up-regulation of PI3K signaling [20, 25], and we found that PI3K is insensitive to formoterol. PI3K stimulates ERK1/2 activation via Raf-1 and Rap-1 in a Ras-independent manner [63, 64] and can counter-balance formoterol-β2AR-cAMP-dependent ERK1/2 inhibition. Indeed, our data show that formoterol markedly inhibits ERK1/2 phosphorylation in chronic hypoxia-exposed PAVSM cells, in which mTOR activation and proliferation occur in a PI3K-independent manner .
Chronic hypoxia-induced proliferation of PAVSM cells requires expression of hypoxia-inducible factor 1 α (HIF1α), which plays a critical role in PAVSM remodeling in human and experimental PH [31, 65]. Notably, ERK1/2 up-regulates HIF1α transcriptional activity via direct phosphorylation that promotes HIF1α nuclear translocation or via regulating binding of HIF1α with its major co-activator p300/cAMP response element-binding protein (CBP) [66–69]. Thus, formoterol may inhibit chronic hypoxia-induced PAVSM cell proliferation via down-regulation of ERK1/2-dependent HIF1α transcriptional activity.
Although much less is known about the regulation of the mTOR signaling pathway by cAMP/PKA, it is shown that cAMP elevation inhibits mTORC1/S6K1 in T lymphocytes , but not in CCL39 fibroblasts  suggesting that effects of cAMP/PKA on mTOR activation are cell type-specific. Our data show that formoterol has no effect on activation of mTORC1 and mTORC2 signaling pathways caused by either PDGF or chronic hypoxia. These data indicate that mitogen- and chronic hypoxia-induced mTOR activation in human PAVSM cells is β2AR-independent.
Taken together, our study demonstrates that formoterol inhibits basal, chronic hypoxia- and thrombin-, but not PDGF-induced human PAVSM cell proliferation potentially via β2AR-dependent inhibition of ERK1/2 signaling pathway; and that anti-proliferative activity of formoterol is provided predominantly by its (R,R) enantiomer. This data suggests that (R,R) formoterol, while having a limited effect as a single agent, may be considered as a potential adjuvant therapy for COPD-associated PH.