Mechanistic basis for the antifibrotic actions of cAMP-based therapies

Abstract

The human and economic impact of idiopathic pulmonary fibrosis and other interstitial lung diseases is enormous, and available therapies are of limited utility. A decade after the introduction of the first antifibrotic agents, two new agents are on the horizon. Nerandomilast is an inhibitor of phosphodiesterase 4B, while treprostinil is an analogue of prostacyclin. Both agents increase intracellular cAMP. Although the smooth muscle relaxant properties of agents that increase cAMP have long been leveraged for the treatment of airway and vascular diseases, potential antifibrotic actions of cAMP elevation are much less well appreciated by clinicians and researchers. The purpose of this review is to discuss the mechanistic underpinnings for a beneficial role of cAMP in fibrotic lung diseases. We briefly review the pathogenesis of fibrotic lung disease, the anatomy of the cAMP pathway, and the myriad ways in which this pathway is disrupted in fibrotic diseases. We then focus on the pleiotropic actions by which cAMP opposes the aberrant phenotypes of immune cells, fibroblasts, and epithelial cells that characterise fibrotic diseases. Finally, we highlight some unanswered questions about, and future opportunities for optimising, therapeutic interventions that leverage the cAMP pathway.

Shareable abstract

Two new pharmacological agents are anticipated to soon become available for treatment of fibrotic lung diseases. Here we review the antifibrotic mechanisms of action attributed to their shared ability to elevate intracellular cAMP. https://bit.ly/4pIMUpY

Introduction

Fibrotic remodelling, a process involving the scarring of normal tissue by the accumulation of extracellular matrix (ECM) proteins such as collagen, affects all organs. It typically reflects the end-stage consequence of a multitude of chronic or repetitive insults [1]. If these are recognised sufficiently early, it may be possible to intervene and mitigate the development of appreciable fibrosis. However, once established, tissue fibrosis is generally irreversible, resulting in enormous morbidity and economic cost [1]. Interstitial lung diseases (ILDs) characterised by pulmonary fibrosis can result from infections (e.g. COVID-19), immune responses (e.g. hypersensitivity pneumonitis or sarcoidosis), toxic exposures (e.g. silicosis or asbestosis) or systemic diseases (e.g. systemic sclerosis). Most commonly, no predisposing cause is identifiable, and it is termed idiopathic pulmonary fibrosis (IPF). Pirfenidone and nintedanib were approved for the treatment of IPF by the United States Food and Drug Administration (FDA) ∼10 years ago, and they have increasingly been implemented in other forms of pulmonary fibrosis. These drugs have been shown to slow progression of pulmonary fibrosis in some patients, but they do not halt progression nor do they reverse established fibrosis. Moreover, their utility is substantially limited by side-effects, predominantly gastrointestinal [2].

Despite the investment of billions of dollars into the development of other potential antifibrotic drugs over the past 20 years, no additional therapies have yet emerged. However, now, two new antifibrotic agents are on the horizon. Nerandomilast, an oral drug developed by Boehringer Ingelheim, was efficacious in separate phase 3 trials for IPF [3] and non-IPF ILD [4], and has been approved recently by the FDA for IPF. Treprostinil, an inhaled agent marketed by United Therapeutics for the treatment of pulmonary arterial hypertension (PAH), has very recently met the primary study end-points in a phase 3 trial for IPF.

Nerandomilast is a selective inhibitor of the phosphodiesterase (PDE) isoform 4B; as such, it inhibits the enzymatic degradation of the second messenger cAMP, thereby elevating intracellular cAMP levels. Treprostinil is a stable analogue of the natural but short-lived prostanoid PGI₂; although it can act via ligation of several receptors, its actions in PAH and probably in pulmonary fibrosis are mainly linked to G protein-coupled receptors (GPCRs), including the PGI₂ receptor IP, that signal via enzymatic generation of cAMP. Pharmacological strategies to elevate intracellular cAMP have long been employed to accomplish smooth muscle relaxation in the bronchi and pulmonary arteries for the treatment of airway diseases (asthma and COPD) and PAH, respectively. By contrast, the basis for an antifibrotic role of cAMP is much less well appreciated by most clinicians and scientists.

The implications of these new clinical trial data for patient management will be discussed by others elsewhere. It should also be noted that recent reviews have addressed either the broad interplay between cAMP and fibrogenesis [5], or the potential of PDE4B inhibition in various ILDs [6, 7]. However, no recent reviews have comprehensively addressed the targeting of both GPCRs and PDEs to leverage cAMP elevation as a shared mechanism for antifibrotic therapeutics. Here, we place the efficacy of these agents in context by reviewing what is known about the cellular and molecular mechanisms through which the cAMP pathway exerts a beneficial role in fibrotic lung diseases, and how exploiting this pathway might be further optimised in the future.

Current concepts of pulmonary fibrosis pathogenesis in brief

It is currently believed that pulmonary fibrosis originates from repeated microscopic injuries to a primed alveolar epithelium, resulting in loss of normal cells and the appearance of aberrant ones [1]. The nature of the injury may be recognisable or idiopathic (i.e. IPF). The stereotypical downstream consequence is an aberrant wound healing response culminating in the pathological activation and expansion of mesenchymal cells: the ultimate cellular effectors of fibrosis [1]. When exposed to pro-fibrotic mediators such as transforming growth factor (TGF)-β elaborated by the injured epithelium or activated macrophages, quiescent fibroblasts undergo phenotypic transdifferentiation into pro-fibrotic or pathogenic fibroblasts, traditionally termed “myofibroblasts”, because they express contractile proteins such as α-smooth muscle actin (SMA) typically associated with smooth muscle cells. Their pathogenic importance derives predominantly from two critical properties: 1) they produce more collagen and other ECM proteins per cell than do fibroblasts; and 2) unlike fibroblasts, they are resistant to apoptosis and therefore accumulate [8–10]. The intrinsic stiffness of fibrotic ECM can reinforce the pro-fibrotic properties of myofibroblasts via a process known as mechanotransduction [11–13]. The ultimate outcome of these processes also reflects aberrant intercellular communication among epithelial cells, mesenchymal cells, and immune cells, most notably macrophages [14, 15].

Anatomy of the cAMP pathway

cAMP signalling has been reviewed comprehensively [16], and is summarised briefly in this section (figure 1). The generation of cAMP is initiated when an extracellular ligand (neurotransmitter, hormone, chemokine, lipid mediator, or drug) binds to a GPCR coupled to a stimulatory G protein α subunit (Gαs). The activated Gαs subunit stimulates membrane-bound adenylyl cyclase to catalyse the conversion of ATP to cAMP. Some well-known Gαs-coupled GPCR ligands include epinephrine and norepinephrine, histamine, serotonin, dopamine, adenosine and prostanoids such as prostaglandin (PG)E₂ and PGI₂. Adenylyl cyclase can instead be inhibited by ligation of GPCRs coupled to an inhibitory G protein α subunit (Gαi), by molecules such as lysophosphatidic acid, endothelin and chemokines. Intracellular levels of cAMP are tightly regulated by its adenylyl cyclase-dependent synthesis along with its PDE-dependent degradation, and possibly through its cellular efflux [17]. There are numerous distinct isoforms and/or splice variants of adenylyl cyclases and PDEs. The expression of individual enzyme species varies by tissue, but most cells express multiple isoforms, albeit with different subcellular localisation. The final key component of the cAMP system is the effector proteins activated by cAMP which ultimately carry out its downstream actions. The canonical effector is cyclic AMP-dependent protein kinase A (PKA), which phosphorylates serine and threonine residues in numerous target proteins, including enzymes such as kinases as well as transcription factors such as cAMP response element-binding protein (CREB). The more recently discovered effector is a guanine nucleotide exchange protein directly activated by cAMP (Epac). Of its two isoforms, Epac-1 is expressed in most cells and tissues, including the lung [18]. PKA and Epac-1 can have distinct, redundant or even opposing actions within a given cell type [19, 20]. Yet another level of regulation involves the spatial sequestration of specific PDE isoforms and effector molecules to A-kinase anchoring proteins that serve as scaffolds to localise the influence of cAMP into discrete signalling hubs or compartments within the cell [21]. This results in spatially discrete cAMP signalling axes which may be differentially influenced by Gαs-coupled GPCR agonists and isoform-specific PDE inhibitors. This mind-boggling degree of molecular and spatial complexity by which both intracellular cAMP levels and its downstream biological actions are regulated is presumed necessary for it to critically influence such an enormous breadth of important cellular functions. Such complexity suggests that there is not a single cAMP axis, but many. The rationale for cAMP-elevating therapies in pulmonary fibrosis is based on an adaptation of Koch's postulates, in which 1) cAMP opposes fibrotic mechanisms in vitro and in vivo and 2) disruption of the cAMP axis is present in pulmonary fibrosis and contributes to pro-fibrotic mechanisms.

FIGURE 1.

Anatomy of the cAMP pathway and potential therapeutic avenues. Intracellular cAMP generation by adenylyl cyclase is determined by the balance of stimulatory and inhibitory inputs governed by ligation of stimulatory G protein α subunit (Gαs)- and inhibitory G protein α subunit (Gαi)-coupled G protein-coupled receptors (GPCRs), respectively. cAMP is negatively regulated by its cleavage by phosphodiesterases (PDEs) into AMP and efflux into the extracellular space (via transporters such as ATP-binding cassette protein C1 (ABCC1)). The effector molecules protein kinase A and exchange protein activated by cAMP (Epac)-1 facilitate downstream signalling cascades, including transcriptional upregulation of antifibrotic molecules such as Kruppel-like factor 4 via cAMP response-element binding protein (CREB) activation. Current therapeutics include activation of Gαs-coupled GPCRs (treprostinil) and inhibition of cAMP breakdown (nerandomilast). AKAP: A-kinase anchoring protein. Figure partially created with BioRender (BioRender.com).

Effect of cAMP in experimental models of pulmonary fibrosis

Pharmacological or genetic manipulation in a variety of animal models reveals that cAMP elevation exerts an antifibrotic action in vivo. For example, cell-permeable dibutyryl cAMP reduced both lung nodules and fibrotic ECM proteins in a chronic rat model of silicosis [22]. In a mouse model of pulmonary fibrosis elicited by targeted injury to alveolar type 2 cells, three distinct PDE4 inhibitors attenuated lung collagen accumulation [23]. Nerandomilast itself has been shown to attenuate fibrosis in the lungs of bleomycin-treated rats [24] and both the lungs and skin of mice subjected to subcutaneous bleomycin to model systemic sclerosis [25].

Both gain and loss of function of Gαs-coupled agonists or their specific receptors have also been shown to modulate fibrosis in animal models. The prostanoids PGE₂ and PGI₂, lipid mediators synthesised from arachidonic acid via cyclooxygenase (COX) enzymes, are well known to regulate numerous cellular functions via cAMP generation. Bleomycin-induced lung fibrosis was indeed shown to be exaggerated in mice treated with the COX inhibitor indomethacin [26], in COX-2-deficient mice [27, 28], in mice with a deficiency of the Gαs-coupled PGI₂ receptor IP [28], and in mice with a deficiency of the Gαs-coupled PGE₂ receptor EP2 [29]. In a gain-of-function approach, potentiation of lung prostanoid levels using a small molecule inhibitor of the prostanoid-degrading enzyme 15-prostaglandin dehydrogenase abrogated the severity of bleomycin-induced fibrosis [30]. A similar outcome was reported with the intrapulmonary administration of the PGI₂ analogue treprostinil, which ligates IP as well as EP2 [31, 32]. Beyond prostanoids, the long-acting β₂-adrenergic receptor (AR) agonist olodaterol [33] and two different agonists of the Gαs-coupled dopamine D1 receptor [34, 35] have also been shown to attenuate mouse models of pulmonary fibrosis.

The capacity for cAMP-acting agents to influence tissue remodelling in the lung extends beyond the parenchyma to the airways and vasculature. A COX-2 inhibitor potentiated airway fibrosis in a mouse model of repeated allergen challenge [36]. Furthermore, mice with a specific deficiency of PGE₂ synthesis, but not wild-type mice, exhibited vascular remodelling in response to allergen challenge, and this was prevented by administration of a PGE₂ analogue [37]. Finally, the antifibrotic actions of cAMP extend to other tissues including the skin [25], heart [38], liver [39] and kidneys [40]. Taken together, these compelling data help to contextualise the clinical trial findings of apparent antifibrotic actions of nerandomilast [3, 4] and treprostinil [41] in pulmonary fibrosis.

Effects of cAMP on key cell types in the pathogenesis of pulmonary fibrosis

To gain a deeper mechanistic understanding of its antifibrotic effects, we review the relevant actions of cAMP in three key cell types central to the pathogenesis and outcomes of pulmonary fibrosis: immune cells, fibroblasts, and epithelial cells. For each cell type, we consider the effects of cAMP on both pathogenic cellular phenotypes (figure 2) and the molecular drivers that underlie them (figure 3).

FIGURE 2.

Effects of cAMP on phenotypes of critical lung cell types. Through activation of stimulatory G protein α subunit (Gαs)-coupled G protein-coupled receptor (GPCRs) by various ligands in macrophages, fibroblasts and alveolar epithelial cells, cAMP signalling orchestrates phenotypic changes in each of these cell types, which mediate antifibrotic actions and could promote fibrosis resolution. cAMP functions to disable macrophages and fibroblasts while promoting the survival and regeneration of alveolar epithelial cells. MF: myofibroblast. Figure partially created with BioRender (BioRender.com).

FIGURE 3.

Pro-fibrotic drivers inhibited by cAMP. cAMP attenuates lung fibrosis by targeting a diverse array of pathways. TGF: transforming growth factor; IL: interleukin; LTB: leukotriene B; ET: endothelin; PI3K: phosphoinositide 3-kinase; JAK: Janus kinase; Erk: extracellular signal-regulated kinase; FAK: focal adhesion kinase; FOXM1: forkhead box protein M1; mTOR: mammalian target of rapamycin; Ca²⁺: calcium; STAT: signal transducer and activator of transcription. Figure partially created with BioRender (BioRender.com).

Immune cells

Even though IPF is not primarily considered an inflammatory disease, macrophages in particular are thought to contribute to disease pathogenesis by virtue of their ability to elaborate pro-fibrotic growth factors, regulate ECM degradation and clearance, and communicate with epithelial cells and mesenchymal cells. By contrast, the pathogenic role of inflammatory cells is more central in non-IPF ILDs triggered by particulates (silicosis), immune complexes (connective tissue diseases), and antigens (sarcoidosis and hypersensitivity pneumonitis).

cAMP has long been recognised to exert anti-inflammatory actions throughout the body, and this potential has motivated the development of PDE inhibitors for a broad spectrum of inflammatory diseases ranging from COPD to psoriasis to inflammatory bowel diseases and beyond [42]. PDE4B and D are the isoforms most highly expressed in immune cells, and it is their inhibition that accounts for most of the anti-inflammatory actions of PDE inhibition [43]. Among Gαs-coupled GPCRs, immune cells generally express the PGI₂ receptor IP, the PGE₂ receptors EP2 and EP4, and the β2-AR, and all of these ligand classes can suppress their activation [44–46]. However, receptor abundance and responsiveness to individual ligands in any cell type can vary depending on anatomical site of origin and phenotypic alterations associated with disease. PGE₂ is one of the most abundant prostanoids and Gαs agonists throughout the body, and it is well known to promote pain, fever and oedema. As such, it is commonly considered a “pro-inflammatory” mediator, and nonsteroidal COX inhibitors of its synthesis are regarded as “anti-inflammatory” agents. This apparent paradox is explained by the fact that these classic effects of PGE₂ reflect its actions on nerves, the hypothalamus and the microvasculature, respectively, rather than on leukocytes. In fact, its direct effects on immune cell functions are overwhelmingly inhibitory and these are consistently mediated by increased intracellular cAMP [46, 47]. Key take-home points from this literature are as follows. First, cAMP-elevating agents suppress the activation of virtually all leukocyte subsets, including macrophages, lymphocytes, neutrophils and eosinophils [44–46]. Second, cAMP inhibits virtually all the functions of each of these activated cell types, including their migration, adhesion, proliferation, generation of reactive oxygen species, and elaboration of inflammatory mediators; the latter reflects inhibition of molecules such as NADPH oxidase, myriad protein kinases, and transcription factors such as NF-κB. Third, cAMP actively promotes a variety of immune-suppressive actions, such as the differentiation of regulatory T-cells and the generation of interleukin (IL)-10 and suppressor of cytokine signalling (SOCS) proteins. However, it should be noted that the effects of cAMP signalling in immune cells is highly nuanced, as it can by contrast also promote distinct inflammatory and fibrogenic processes by enhancing M2 polarisation [48, 49] as well as IL-6 generation [50].

Fibroblasts

In view of their central role in elaborating ECM, the impact of cAMP on fibroblasts and myofibroblasts is vital. Fibroblasts express a wide array of Gαs-coupled receptors. Those that are expressed at high levels include IP, EP2, EP4 and β₂-AR. An extensive body of literature demonstrates that PGE₂ (via EP2>EP4) suppresses virtually all the pathogenic functions of normal lung fibroblasts activated in vitro (reviewed in [51]). Many of these actions are shared by PGI₂ [52], β₂-AR agonists [53] and PDE inhibitors [54]. Specific effects include inhibition of their proliferation [55], chemotaxis [56, 57], synthesis of collagen and other ECM proteins [55], lysyl oxidase-mediated collagen cross-linking [58] and differentiation into α-SMA-expressing myofibroblasts [59, 60]. At the same time, elevation of cAMP promotes fibroblast apoptosis [61] and collagen degradation [62]. These suppressive actions of cAMP largely extend to lung fibroblasts obtained from patients with IPF and other fibrotic disorders as well. The breadth of suppressive actions of cAMP on fibroblast functions is indeed remarkable, and reflects the fact that cAMP opposes or inhibits numerous cellular processes, signalling pathways, and mediators implicated in pulmonary fibrosis. Fundamental cellular processes influenced by cAMP include transcription, translation [63], epigenetic control [64], and the activity of kinases and phosphatases. A sample of relevant targets whose actions are regulated by cAMP include pro-fibrotic mediators TGF-β, IL-13, endothelin-1, complement, lysophosphatidic acid, various mitogenic growth factors, and matrix stiffness itself; intracellular calcium; kinases including the mitogen-activated protein (MAP) kinase p38 and Erk, phosphoinositide 3 kinase/Akt, Janus kinase, and focal adhesion kinase; transcription factors forkhead box protein M1, NF-κB and signal transducer and activator of transcription; transcriptional co-activators YAP/TAZ; the Wnt/β-catenin pathway; and the translational controller mammalian target of rapamycin [24, 63, 65–70]. Many of these regulatory effects reflect the phosphorylation of target proteins by PKA. Phosphorylation-dependent activation of CREB also accounts for the ability of cAMP to increase the production of an array of antifibrotic molecules, including the transcription factors Kruppel-like factor 4 (KLF4) and peroxisome proliferator-activated receptor-γ, the phosphatases PTEN and MAP kinase phosphatase 1 (MKP1), SOCS proteins, and the decoy receptor IL-13Rα2 [71–75]. In some instances, Epac-1 has been implicated in the antifibrotic actions of cAMP on fibroblasts [19, 76].

Importantly, PGE₂ does not merely prevent myofibroblast differentiation when added before or simultaneously with TGF-β, but it can substantially reverse the phenotype of already established myofibroblasts [77]. This is reflected by reduced expression of α-SMA and its organisation into stress fibres, as well as of collagen and a panoply of other fibrotic genes. Such “dedifferentiation” is accompanied by global alterations in the fibroblast transcriptome involving thousands of genes [70, 78]. Dedifferentiation has also been observed with other cAMP-elevating agents including both a nonselective PDE4 inhibitor [79] as well as nerandomilast itself [80], a specific EP2 agonist, and a direct activator of adenylyl cyclase [70], and in myofibroblasts obtained from patients with systemic sclerosis [79]. A notable consequence of such dedifferentiation is the reacquisition of sensitivity to apoptosis [70]. This phenomenon is increasingly recognised to be necessary for the clearance of pathogenic myofibroblasts, which is typically observed in young mice exposed to single-dose bleomycin [81], but it is absent in aged mice in which pulmonary fibrosis is persistent [82]. Interestingly, the capacity to elicit dedifferentiation is also shared by two antifibrotic molecules whose expression is induced by cAMP, namely KLF4 [71] and MKP1 [72]. Finally, it is noteworthy that this ability of cAMP elevation to promote myofibroblast dedifferentiation is not shared by the FDA-approved antifibrotic agents pirfenidone and nintedanib [72].

Epithelial cells

An intact alveolar epithelial barrier protects the lung against both entry of inhaled toxic materials and leak of vascular contents, but is also essential to promote repair and regeneration following injury. Indeed, loss of normal alveolar and distal airway epithelial cells as well as their replacement by aberrant cell phenotypes are pivotal events in the initiation and progression of fibrosis. Under normal conditions, PGE₂ is the predominant Gαs ligand produced by alveolar epithelial cells (AECs) [83], and the autocrine and paracrine actions of epithelial-derived PGE₂ with subsequent cAMP signalling play key roles in these homeostatic and antifibrotic properties. For example, the ability of AECs to suppress fibroblast proliferation in response to mitogens [84, 85] and myofibroblast differentiation in response to TGF-β [86] depends on their elaboration of PGE₂. Both endogenous and exogenous PGE₂ were shown to promote wound closure and migration of airway epithelial cells in culture [87]. A recent study identified PGE₂ and PGI₂ as the most promising receptor ligands to emerge from an extensive transcriptomic screen of potential agents capable of promoting lung epithelial cell regeneration in an organoid model of cigarette smoke exposure [88]. AEC expression of β₂-AR is abundant [89], and the ability of its agonists to promote alveolar fluid clearance [90] is well known. However, their influence on epithelial cell migration and survival are under-studied, and in the aforementioned cigarette smoke exposure study [88], the ability of olodaterol to promote epithelial regeneration was limited. Salutary actions of PGE₂ on epithelial integrity extend to its ability to prevent apoptosis [91]. As noted previously, PGE₂ promotes apoptosis in fibroblasts [61, 91]. The contrasting actions of PGE₂ on the survival of these two critical cell types (along with impaired PGE₂ production in IPF, described later) contributes to the long-recognised “apoptosis paradox” thought to be pivotal in the pathogenesis of IPF [91]. Finally, nerandomilast has recently been shown to inhibit production by cytokine cocktail-treated small airway human epithelial cells of a number of fibrosis-associated biomarker proteins, including matrix metalloproteinase-7, osteopontin and connective tissue growth factor [24]. It is evident, then, that prostanoids and cAMP signalling exert broad actions which both suppress the pathogenic and pro-fibrotic functions while promoting the homeostatic, regenerative, and antifibrotic functions of lung epithelium.

Disruptions to the cAMP axis in fibrotic lung disease

States of disease typically reflect not only the actions of pathogenic factors, but also the loss of those responsible for homeostasis. A well-known example is the contribution in cancer of the downregulation of tumour suppressors complementing and synergising with the actions of oncogenes. In analogous fashion, the importance of the cAMP axis as an antifibrotic brake is strongly suggested by a sizeable body of literature documenting its multifocal impairment in pulmonary fibrosis. The first report of such a perturbation showed that lung lavage levels of PGE₂ were reduced in patients with IPF as compared to normal controls [92]. It was subsequently found that lung fibroblasts isolated from IPF patients exhibited an impaired capacity to synthesise PGE₂ owing to impaired expression of COX-2 [93], and such a defect was later extended to the synthesis of PGI₂ [94]. ECM stiffness, which reinforces myofibroblast phenotype in fibrotic tissue, has likewise been shown to restrain PGE₂ synthetic capacity in fibroblasts owing to impaired expression of COX-2 as well as the distal enzyme PGE synthase [95, 96]. Tissue prostanoid levels are also determined by degradation via the enzyme 15-prostaglandin dehydrogenase, and its increased expression in the lungs of IPF patients further reduces PGE₂ levels [97]. Expression of the key cAMP-elevating PGE₂ receptor, EP2, has been reported to be reduced in lung fibroblasts in some studies of patients with IPF [98] and bleomycin-treated mice [29]; such receptor changes have generally been paralleled by dampened antifibrotic responses to exogenous PGE₂. Differentiation of fibroblasts to myofibroblasts with TGF-β has itself been shown to downregulate gene expression of a variety of Gαs-coupled receptors, including prostanoid receptors EP2, EP4 and IP, the β₂-AR and adenosine receptors 2A and 2B [99]. One possible unifying mechanism to explain reduced gene expression of both the COX-2 enzyme [100] and the EP2 receptor [101] in fibrotic fibroblasts is DNA hypermethylation. Upregulated expression of fibroblast PDEs in response to EP2 ligation [102] and in myofibroblasts from fibrotic tissues [39, 103] may also contribute to Gαs desensitisation. With respect to downstream cAMP effectors, fibroblasts from some patients with IPF have been reported to exhibit specific impairments in PKA activation [104], while TGF-β treatment has been shown to diminish Epac-1 expression [105]. Finally, expression of secondary cAMP-dependent antifibrotic molecules KLF4 [71] and MKP1 [72] was likewise reduced in lung fibroblasts from IPF patients. Of note, genetic deletion of many of these antifibrotic cAMP axis components downregulated in patients has been shown to exacerbate experimental fibrosis [27–29] and in some instances, to impair its spontaneous resolution [71, 72]. The breadth of these defects strongly suggests that downregulation of the cAMP axis not only predisposes to fibrogenesis, but may actually be necessary for pulmonary fibrosis to occur.

Unanswered questions and future considerations

A number of uncertainties and open questions regarding cAMP-elevating therapeutics remain, and addressing these in future studies will be essential if we are to realise their potential. Some of these questions are considered briefly in this section.

Targeting various components of the cAMP axis

cAMP elevation can be accomplished by interventions directed at a number of the axis components depicted in figure 1. Conventional strategies include administration of GPCR agonists or PDE inhibitors, exemplified by treprostinil and nerandomilast, respectively. A combination of both of these is also readily envisioned. These approaches are discussed herein. Other strategies which have received less attention, and which are not discussed further include administration of 1) a direct adenylyl cyclase activator [106]; 2) analogues of cAMP that are cell-permeable and possibly PDE-resistant [107]; 3) analogues of cAMP that selectively activate either PKA or Epac-1 [19]; 4) inhibitors of the efflux pump ATP-binding cassette protein C1 [108]; and 5) inhibitors of 15-prostaglandin dehydrogenase [109]. Our understanding of the relative efficacy of all of these various approaches remains limited, even in reductionist cell culture models, and is virtually nonexistent in animal models and in patients.

EP2, EP4 and IP are among the numerous Gαs-coupled receptors which are candidates for agonism. Their cellular distribution in ILDs as well as their relative importance and advantages remain incompletely understood. Again, studies using cell-specific transgenic mice may help to illuminate their importance. Although much of what we know about the antifibrotic actions of cAMP elevation derive from experiments with PGE₂ itself, it is a less desirable therapeutic option because of its ability to also ligate potentially pro-fibrotic receptors EP1 and EP3. Of note, treprostinil ligates not only IP, but also the Gαs-coupled receptors EP2 and the prostaglandin D₂ receptor DP1 as well as the non-GPCR nuclear receptors peroxisome proliferator-activated receptors α and β [31, 32, 110]. The relative roles of these receptors in mediating its antifibrotic actions remain unclear, as is whether its concomitant EP2 agonism offers an advantage over a pure IP agonist such as selexipag. It is also necessary to acknowledge the uncertainty surrounding the actions of β₂-AR agonists in fibrosis. On the one hand, the β₂-AR is present in all the pertinent cell types discussed, and its agonists have demonstrated antifibrotic actions in vivo [33] and in fibroblasts in vitro [33, 111]. On the other, this class of agonists has been extensively employed for decades in patients with respiratory diseases including ILDs, and there is little evidence to suggest an antifibrotic signal. A final issue surrounding GPCRs that is deserving of discussion involves the question of receptor downregulation. A generic facet of this is the phenomenon known as “desensitisation” in which the strength of signalling following ligation of certain GPCRs diminishes on repeated stimulation, owing to mechanisms including β-arrestin-mediated inhibition and receptor internalisation [112]. A more specific aspect of this phenomenon is that referenced earlier, in which gene expression of a variety of antifibrotic Gαs-coupled receptors is variably observed to be downregulated in association with pulmonary fibrosis or models thereof [29, 98, 99]. This phenomenon raises the question of “resistance” to specific GPCR agonists in specific patients, and if this should influence the selection of such agonists as therapeutics. It seems likely that various resistance-inducing molecular defects may exist in different cells or regions of the lung, or may emerge over time in a given patient with or without treatment. As these would be difficult to ascertain diagnostically, we favour the more practical strategy of employing agents that target multiple components of the cAMP axis, either simultaneously or sequentially.

Although the relative efficacy of nerandomilast versus treprostinil may be determined by future clinical experience and/or trials, a specific generic question of interest is whether a GPCR agonist or a PDE inhibitor is likely to be superior. Certainly, each approach is likely to have its advantages and disadvantages. However, it is worth noting that in a lung organoid model, PDE inhibition alone failed to promote epithelial regeneration following challenge with cigarette smoke, unlike prostanoids [88]. Likewise, the ability of nerandomilast to dedifferentiate myofibroblasts and modulate the transcriptome was substantially less than that of PGE₂ alone [80]. These findings are concordant with experience in treating PAH, in which some patients already on the cyclic GMP-specific PDE5 inhibitor sildenafil benefitted from switching to the direct guanylate cyclase stimulator riociguat [113]. Such data suggest that stimulating production of a cyclic nucleotide may, under certain circumstances, be more efficacious than inhibiting its degradation.

The “bathtub” analogy (figure 4) helps to frame the respective roles of Gαs-coupled GPCR agonism and PDE inhibition as determinants of intracellular cAMP levels. Because merely “plugging the drain” is insufficient to fill the cAMP “bathtub”, the clinical success of PDE4B inhibition by nerandomilast implies that the cAMP “faucet” is at least partially open. Potential endogenous limitations to cAMP production in pulmonary fibrosis and how this might be pharmacologically restored is worthy of further consideration. Herein lies the potential advantage of combining an agent that opens the faucet with one that blocks the drain, with treprostinil plus nerandomilast being the obvious combination that can be envisioned in the relative short-term. While such additivity has frequently been observed in a variety of cell types [114, 115], there are also examples in which cAMP levels fail to correlate directly with functional effects [116]. This may be because considering a cell to be a single homogenous solution of molecules capable of unfettered interactions is an outmoded oversimplification which ignores the importance of spatial segregation of cAMP signalling bestowed by localisation of specific GPCRs and PDE isoforms. Nonetheless, the faucet-and-drain analogy may still hold relevance when combing a GPCR agonist and PDE inhibitor for any given individual subcellular domain. Another potential advantage of combining PDE inhibition with GPCR activation derives from observations that increased expression of PDEs may contribute to desensitisation of certain GPCRs [102]. Finally, a PDE inhibitor might increase the duration of the cAMP signal arising from GPCR ligation, reducing the required frequency of administration of the latter. Of course, as exemplified by the hypotensive risk of combining cGMP-acting nitrates and PDE5 inhibitors [117], the possibility of adverse consequences from employing cAMP faucet openers together with drain blockers will be an important consideration as dosing alternatives are evaluated going forward.

FIGURE 4.

Conceptual schematic depicting determinants of intracellular cAMP concentration. Regulation of intracellular (or intracompartmental) cAMP levels is analogous to that determining the water level in a bathtub: the balance between faucet flow (G protein-coupled receptor (GPCR)-mediated cAMP generation) and drain flow (cAMP degradation or cellular egress). Nerandomilast functions to elevate intracellular cAMP levels by “plugging the drain”, while treprostinil increases cAMP by “opening the faucet”. It therefore follows that stimulatory G protein α subunit (Gαs)-coupled GPCR agonists or inhibitory G protein α subunit (Gαi)-coupled GPCR inhibitors would “open the faucet” and likewise increase intracellular cAMP. +: factors that elevate cAMP levels; −: factors that reduce cAMP levels; [cAMP]_i: intracellular cAMP concentration; PDE: phosphodiesterase. Figure partially created with BioRender (BioRender.com).

Leveraging the pleiotropic actions of cAMP

While its enormous breadth of antifibrotic actions is an attractive feature of the cAMP pathway, our ability to optimally leverage it therapeutically might benefit from a clearer understanding of the importance of specific cellular (immune cell versus epithelial cell versus fibroblast) and mechanistic (promoting myofibroblast apoptosis versus ECM clearance) targets. Employing a loss-of-function approach (e.g. using cell-specific gene knockout) in combination with administration of specific cAMP-elevating agents can provide a powerful means of interrogating the importance of individual cellular and mechanistic targets. The importance of individual targets of course might also differ in various types of patients. As an obvious example, targeting immune cells may be more beneficial in patients with recognised underlying inflammatory processes than in patients with IPF.

Personalising cAMP-elevating therapies

Are there certain patients who might benefit the most from cAMP-elevating therapy? One example might be older patients at risk of IPF who are on medications such as corticosteroids and nonsteroidal anti-inflammatory agents which inhibit endogenous prostanoid generation. Heterogeneity in cAMP signalling is of course plausible within any group of patients regardless of specific ILD diagnosis. The ability to employ a more sophisticated personalised medicine approach would be facilitated if we could develop the means to estimate the overall lung cAMP status of individual patients. This might be accomplished, for example, using a plasma or lung lavage biomarker, or an imaging modality, which determined levels of cAMP itself or of its downstream surrogate, phospho-CREB.

Conclusions

Since its discovery in 1957, research into cAMP has garnered a total of four Nobel Prizes and provided new insights into receptor biology, G proteins, and intracellular signalling cascades, as well as physiology, pathology and therapeutics. Recent positive clinical trial data employing two different strategies for elevating cAMP in pulmonary fibrosis [3, 4, 41] builds upon a substantial body of research conducted over the past several decades. Standout themes from this work include the remarkable degree of pleiotropy by which cAMP regulates the relevant phenotypes and behaviours of many critical cell types, and the remarkable number of defects in cAMP generation and effector machinery that characterise tissue fibrosis.

Reaching the “holy grail” in pulmonary fibrosis therapeutics requires promoting resolution of established tissue remodelling, rather than merely inhibiting initiation or progression. This requires enhancing clearance of aberrant cells and ECM as well as regeneration of functionally normal cells to permit functional recovery [118]. The currently approved antifibrotic agents pirfenidone and nintedanib are incapable of accomplishing this. Despite the broad and deep antifibrotic actions of cAMP-elevating agents and their potential to enhance cellular phenotypes favouring fibrosis resolution in vitro and in mouse models, it remains to be determined if therapeutic agents acting on this pathway can promote fibrosis resolution in patients. Future research should strive to determine if and how optimising the potential of cAMP-based strategies, perhaps in combination with other strategies, can accomplish this ultimate therapeutic objective.

Questions for future research

Molecular and cellular biology

• How does the spatial segregation of cAMP signalling within lung cell types influence its antifibrotic actions?

• What downstream molecular and cellular targets of cAMP are most important in mediating its antifibrotic effects within fibroblasts, immune cells, and epithelial cells?

• What signalling impairments in the cAMP axis predispose individuals to IPF and progressive pulmonary fibrosis, and how do they affect cellular responses to endogenous ligands and cAMP-based therapies?

Therapeutic considerations

• What are the relative antifibrotic contributions of cAMP-boosting PDE inhibitors and GPCR agonists in IPF and progressive pulmonary fibrosis in general?

• Can nerandomilast and treprostinil combination therapy additively or synergistically augment their antifibrotic actions?

• Might alternative strategies such as antagonising cAMP efflux pumps or GPCRs that inhibit cAMP production also have therapeutic potential?

• Can patient heterogeneity in cAMP signalling capacity be leveraged for precision approaches to therapy in IPF and progressive pulmonary fibrosis?