Emerging concepts in smooth muscle contributions to airway structure and function: implications for health and disease

Abstract

Airway structure and function are key aspects of normal lung development, growth, and aging, as well as of lung responses to the environment and the pathophysiology of important diseases such as asthma, chronic obstructive pulmonary disease, and fibrosis. In this regard, the contributions of airway smooth muscle (ASM) are both functional, in the context of airway contractility and relaxation, as well as synthetic, involving production and modulation of extracellular components, modulation of the local immune environment, cellular contribution to airway structure, and, finally, interactions with other airway cell types such as epithelium, fibroblasts, and nerves. These ASM contributions are now found to be critical in airway hyperresponsiveness and remodeling that occur in lung diseases. This review emphasizes established and recent discoveries that underline the central role of ASM and sets the stage for future research toward understanding how ASM plays a central role by being both upstream and downstream in the many interactive processes that determine airway structure and function in health and disease.

airway structure and function are important for respiratory and pulmonary physiology across all ages. Indeed, a number of normal and pathophysiological processes influence the initial embryonic formation and differentiation of cell types in conducting airways, as well as their function or dysfunction throughout life. Here, such processes are further modulated by interactions between cells, between cells and the environment, and, importantly, the mechanical forces of breathing. In these contexts, dysfunctional, typically excessive, narrowing of the conducting airways, and impaired relaxation occur in clinically important diseases such as asthma across age groups, in bronchitis, and in chronic obstructive pulmonary disease (COPD). Such functional changes may be accompanied or exacerbated by concomitant structural changes involving thickening of airway layers (particularly bronchial epithelium, also frequently dysfunctional) and airway smooth muscle (ASM) and by varying degrees of fibrosis. Etiologies for airway structural and functional changes vary with age, context, and exposure but include developmental abnormalities (e.g., genetic disorders, maternal and fetal insults), allergens and infectious agents, environmental exposures (e.g., cigarette smoke, toxins, and pollutants), and intrinsic factors such as age and sex (Fig. 1). Here, not only is it important to understand the complex molecular, genetic, proteomic, and physiological processes within a cell type (epithelial cells, ASM, fibroblasts, airway nerves, resident and circulating cells of the immune system, etc.) and their perturbation in disease, but also 1) how interactions between cell types contribute to mutual changes in cellular structure/function and to overall airway functionality or dysfunction; 2) the potential contribution of the extracellular matrix (ECM) that airway cells reside in and interact with; and 3) the role of mechanical forces exerted by breathing. Thus understanding the myriad of factors that drive normal airway structure and function from embryonic lung formation onward in cell- and context-specific fashions is understandably challenging (despite the many large-scale, high-throughput technologies) yet necessary toward appreciating how intrinsic and external forces drive induction, maintenance, exacerbation, and (where possible) alleviation and resolution of lung disease.

Fig. 1.

Airway development and growth across the ages in the context of disease. A multitude of intrinsic and extrinsic processes contribute to the structure and function of the bronchial airways at different life stages. In utero lung development involves intrinsic processes such as genetics, maternal and fetal steroids, mechanical forces induced by fetal breathing and external pressures, and, in the context of perinatal disease, immune and infectious processes. Postnatally, except for steroids, such processes can continue to contribute to airway growth or its disruption, especially in the context of prematurity and iatrogenic processes such as mechanical ventilation, infection, etc. With progressive development and aging, normal processes such as mechanical forces of breathing, cell-cell and cell-matrix interactions, aging and cellular senescence mechanisms, diet-induced changes, etc., contribute to airway growth, its maintenance or normal aging-related changes, modulated by sex steroids during different life stages. Exposure of the normal airway to insults such as allergens, microbes, or viruses or to environmental factors (pollutants, tobacco smoke) are overlaid on these normal processes to contribute to disease. Figure was generated using ScienceSlides graphics from Visiscience.

While the relative importance of different cell types in the airway can be argued, from a functional standpoint, ASM plays a critical role in regulating airway tone and contractility, which represent a balance between contractile vs. dilatory processes in response to local or circulating factors, i.e., processes and factors that contribute to the airway hyperreactivity (AHR) and impaired bronchodilation in diseases such as asthma. From a structural standpoint, extrinsic and intrinsic factors can contribute to ASM cell hyperplasia and/or hypertrophy, also resulting in reduced airway lumen. Furthermore, fibrotic processes can contribute to altered mechanical properties of the airway (stiffening) with implications for the work of breathing, and for the functional efficacy of ASM. All of these interconnected processes play variable roles in different lung diseases such as asthma, COPD, and pulmonary fibrosis. Accordingly, understanding the role of ASM per se in the airway becomes key. In this regard, the mechanisms that regulate the important aspect of ASM, i.e., contractility, in response of extrinsic stimuli of airway innervation, secreted factors from other airway cell types (epithelium, fibroblasts, immune cells, and vasculature), and/or circulating mediators still remain in the discovery phase and represent an exciting aspect of basic, translational, and even clinical lung research. Here, the idea that ASM is more than a passive recipient of external signals (regardless of source) and actively contributes to modulation of other cell types (and ASM itself), inflammation and its resolution, and the overall changes in airway structure and function in the context of disease makes for an exciting landscape for investigation.

In this review, I highlight, by no means in a comprehensive manner, recent themes and discoveries that have contributed to furthering the idea of ASM as more than a passive recipient and responder to external signals. I begin with novel findings relating to the unquestionable major role for ASM, viz. contractility involving intracellular Ca²⁺ concentration ([Ca²⁺]_i) and Ca²⁺ sensitization, expounding on the role of G protein-coupled receptor (GPCR) based and non-GPCR-based signaling mechanisms. I next address emerging roles for ASM beyond contractility in airway disease via its contributions to remodeling: cell hypertrophy and proliferation, extracellular matrix formation, generation of mediators and growth factors. More complex and relatively underexplored topics are then addressed including epigenetic mechanisms, noncanonical roles of mitochondria, the increasing recognition of sex differences and sex steroids, and limited exploration of ASM in perinatal airway development and growth. These advances not only provide insight into ASM biology and disease pathophysiology but also point to novel approaches and targets for treatment of lung diseases.

Novel Regulatory Pathways in Intracellular Ca²⁺ and Contractility

The fundamental mechanisms involved in regulation of baseline and agonist-induced elevation of [Ca²⁺]_i in ASM have been largely established (251, 260, 282, 335, 405, 417, 454, 470, 483, 497, 563). Briefly, bronchoconstrictor agonists, acting via G_q-coupled receptors elevate [Ca²⁺]_i through several pathways that involve Ca²⁺ influx and sarcoplasmic reticulum (SR) Ca²⁺ release. Here, production of the second messengers inositol trisphosphate (IP₃) and cyclic ADP ribose leads to intracellular Ca²⁺ release via SR channels that are activated by these second messengers (IP₃-sensitive and ryanodine receptor channels, respectively) (Fig. 2).

Fig. 2.

Regulation intracellular Ca²⁺ ([Ca²⁺]_i) in airway smooth muscle (ASM). While a number of mechanisms are well recognized, including agonist-induced production of the second messengers IP₃ (via the enzyme phospholipase C; PLC) and cyclic ADP ribose (cADPR) via the ectoenzyme CD38, leading to sarcoplasmic reticulum Ca²⁺ release from IP₃ receptor and ryanodine receptor channels, respectively, and Ca²⁺ influx through various types of channels, there are new regulatory pathways recently reported. For example, PLC can be upregulated by PKA and PKG but inhibited by flavanols and PDE4, while IP₃ can regulate Ca²⁺ sparks and influx through voltage-gated Ca²⁺ influx channels (VGCC). Calcineurin (usually associated with downstream transcription) can regulate RyR channels and in turn be regulated by TRP influx channels. Influx of extracellular Ca²⁺ ([Ca²⁺]_o) can occur through VGCCs, TRPC, and TRPV channels as well as reverse mode Na⁺/Ca²⁺ exchange (NCX). In terms of force sensitization involving RhoA, active RhoA levels can be regulated by RhoGAPs and RhoGEFs, an emerging area in ASM. CaCC, calcium-activated chloride channel; NAD, nicotinamide adenine dinucleotide; BKCa, big calcium-activated potassium channel; MLCP, myosin light chain phosphatase; IP3R, IP3 receptor; SERCA, sarcoendoplasmic reticulum calcium ATPase; SR, sarcoplasmic reticulum.

In terms of Ca²⁺ influx and efflux, a number of mechanisms have been described, although their roles under baseline (resting) conditions, in the context of agonist stimulation, and in pathophysiology are variable depending on the study (223, 243, 244, 246, 319; elegantly reviewed for example in Ref. 223). Key mechanisms include voltage-gated Ca²⁺ channels (VGCCs) (260, 405, 454, 483) including L-type and T-type channels, receptor-operated channels, nonselective cation channels particularly transient receptor potential channels (TRPs), Na⁺/Ca²⁺ exchange (9, 10, 224, 318, 424, 458), and, importantly, store-operated Ca²⁺ influx channels, the latter being activated upon SR Ca²⁺ depletion. Some recent developments regarding specific influx pathways are described further below (summarized in Fig. 2). The role of Na⁺/Ca²⁺ exchange (NCX), particularly in the context of its influx mode, is not clear for ASM. While the NCX protein is expressed (and even upregulated in airway inflammation for example), whether it operates largely in the efflux mode to extrude [Ca²⁺]_i or whether it can bring in Ca²⁺ under certain conditions remains to be determined. Here, an interesting question may be what role extracellular Ca²⁺ ([Ca²⁺]_o) plays in modulating influx (or even efflux) pathways in ASM. Unlike in cardiac muscle, where increasing [Ca²⁺]_o is readily associated with increasing [Ca²⁺]_i and contractility, this relationship is less well defined in ASM. Nonetheless, [Ca²⁺]_o is certainly important for influx of Ca²⁺ via TRPs, reverse NCX, store-operated calcium entry (SOCE), and, as recently recognized, regulating the Ca²⁺-sensing receptor (CaSR; described in more detail below).

While this review is not specifically focused on exploring Ca²⁺ influx/efflux pathways, per se, in the context of recent discoveries regarding mechanisms regulating ASM contractility and of novel bronchodilators it is important to mention the contribution of membrane potential. For example, T-type VGCCs can increase force (223, 244), whereas blocking K⁺ channels (which normally induce membrane hyperpolarization and reduced [Ca²⁺]_i) results in contraction (243). Furthermore, Cl⁻ movement in ASM may also modulate contractility vs. relaxation (242), wherein elevated [Ca²⁺]_i for example in response to agonist stimulation can activate small-conductance Cl⁻ channels and further cause depolarization. Chloride may further be relevant to the potential role of γ-aminobutyric acid (GABA) in ASM (see below). In the context of bronchodilation, K⁺ channels are known to cause membrane hyperpolarization (69).

Beyond Ca²⁺, activation of the myosin light chain (MLC) kinase (MLCK)-MLC-actin/myosin cascade leads to contraction, while Ca²⁺ sensitization occurs through the RhoA and Rho kinase (ROCK) mechanism. Here, membrane potential itself can also regulate Ca²⁺ sensitization via ROCK (319). Accordingly, mechanisms that directly or indirectly modulate the Ca²⁺ and force regulatory cascades in ASM have the potential to enhance contractility (e.g., in the context of AHR in asthma), suppress it in the context of bronchodilation, or, conversely, impair bronchodilation itself. While this review cannot comprehensively cover the many such modulatory mechanisms known, some recent insights into Ca²⁺ regulation in ASM may be particularly relevant to understanding disease pathways.

Even with well-established pathways for Ca²⁺ and force regulation, there have been recent developments. For example, miRNAs and transcription factors have been shown to regulate the second messenger CD38 (138, 197, 259). Modulation of the enzyme phospholipase C (PLC) by several upstream mechanisms such as protein kinases A and G (PKA and PKG) (373), flavanols in the context of bronchodilation (68), and phosphodiesterase-4 (PDE4) in the context of quercetin effects (525) could contribute to altered Ca²⁺ regulation (Fig. 2). IP₃ has been shown to activate Ca²⁺ influx in ASM (490) and regulate local Ca²⁺ sparks (323), while ryanodine receptor (RyR) sensitization results in abnormal Ca²⁺ handling (113). In terms of Ca²⁺ sensitization, ROCK appears to be important in mediating AHR in the context of environmental exposure and obesity (265, 298), while regulation of RhoA activation [via RhoGEFs (guanine nucleotide exchange factors) and RhoGAPs (GTPase activating proteins)] is an emerging (but thoroughly underexplored) area in ASM biology (46).

GPCRs.

Major bronchoconstricting agonists such as ACh, histamine, and endothelin-1 work through GPCRs, a superfamily of plasma membrane proteins that transduce extracellular signals not only to elevate (or decrease) [Ca²⁺]_i but also to trigger other intracellular cascades important for cellular function. While common aspects of GPCR-mediated increase in [Ca²⁺]_i are long recognized (as summarized above), some more recent aspects have also been noted, in the context of membrane potential. For example, muscarinic GPCRs can induce changes in membrane potential (40) while, conversely, membrane potential can modulate GPCR activity (41, 322, 333).

GPCRs (particularly those on ASM) continue to be the major drug targets for diseases such as asthma, allergy, and COPD, with the well-studied G_q-coupled pathway (which activates PLC-IP₃, thus elevating Ca²⁺) being key. Conversely, the also-well-studied G_s-coupled pathway (which increases cAMP) is critical for bronchodilation, particularly through β₂-adrenoceptor action. While Ca²⁺ can be indirectly increased through G_i-coupled pathway (which reduces cAMP), this aspect of GPCR signaling in ASM is less understood (49, 134, 398, 402, 403, 563).

One novel bronchodilatory mechanism that continues to generate excitement (and intrigue) in the field is bitter taste receptor (BTR)-induced effects (Fig. 3). Originally identified as acting through the TAS2R family of GPCRs to elevate [Ca²⁺]_i yet induce bronchodilation (135), studies have attempted to further explore this pathway and explain the dichotomy (13, 30, 76, 420, 472, 473, 548, 589, 591). Interest in the biology of BTRs in the airway is enhanced by recent findings of genetic variants in the context of infection, rhinosinusitis, and asthma (472, 561, 582). Given the diversity of [Ca²⁺]_i regulatory pathways in ASM, there is potential for BTRs to activate or inhibit these depending on agonist, context, and perhaps species. Here, the point of interest may be the fact that TAS2Rs show low specificity and affinity to a wide range of bitter compounds, thus allowing for a diverse portfolio of signaling, depending on the agonist, concentration, whether the receptor desensitizes (437), and perhaps even restricted domains of signaling. Accordingly, studies have found that BTRs can induce membrane hyperpolarization via iberiotoxin-sensitive Ca²⁺-activated K channels (135, 420) and nonselective cation channels (591) and interact differently with specific bronchoconstrictors (420) (e.g., denatonium interacts with muscarinic receptors, while chloroquine is more broad). Furthermore, there is the emerging idea that BTRs can activate different Ca²⁺ signaling pathways in context-specific fashions. For example, one group has suggested that TAS2R stimulation activates Gβγ at baseline conditions to increase [Ca²⁺]_i, while blocking activated VGCCs under conditions of bronchoconstrictor agonist stimulation to induce bronchodilation (589) (Fig. 3). More recently, another group has used the differential effects of specific TAS2R agonists to explore relationships between bitter tastants vs. bronchoconstrictor GPCR agonists in human ASM cells (76). They found that the heterogeneity in the inhibitory responses of TAS2R hinges on the bronchoconstrictor used [e.g., chloroquine inhibits histamine effects, but not endothelin, different from the findings of another group (420)], rather than the TAS2R subtype or even the G_q or G_i coupling of the bronchoconstrictor GPCR. Thus there could be a low-efficiency [Ca²⁺]_i stimulation [also suggested by other groups (589)] and a higher efficiency inhibition of [Ca²⁺]_i when specific constricting agonists are present. Separately, the bronchodilating effects of BTRs may rely on interactions with β₂-adrenoceptor expression and signaling (13, 272, 548). For example, one study showed that TAS2R activation can induce relaxation even in the face of adrenoceptor desensitization (13) (making it an exciting alternative for bronchodilation in asthmatic subjects desensitized to β-agonists), while a more recent study suggests that β₂-adrenoceptors may act as a double-edged sword: they increase cell surface expression of TAS2Rs (thus enhancing their function) but may also induce TAS2R desensitization when β-agonists are present (272), consistent with previously observed modest desensitization in human ASM and monkey airways (437). Whether the extent of desensitization depends on TAS2R subtype in ASM remains to be determined.

Fig. 3.

Bitter tastant receptor (BTR) in ASM. There is continuing interest and development regarding this G protein-coupled receptor (GPCR) based signaling mechanisms in ASM. Working through a G_q-coupled mechanism, TAS2R activation can increase SR Ca²⁺ release via IP₃ receptor channels, but intriguingly causes bronchodilation. This latter effect could involve activation of Ca²⁺-activated K⁺ channels (BK_Ca) and chloride channels, but also inhibition of Ca²⁺ sensitization and actin polymerization. Recent findings suggest that BTRs can also counteract muscarinic receptor enhancement of Ca²⁺ influx via VGCCs, suggesting that under baseline conditions BTRs may enhance [Ca²⁺]_i but, in the presence of bronchoconstrictor agonist, act to reduce [Ca²⁺]_i. Furthermore, BTRs appear to have antimitogenic properties. Separately, BTRs can interact with β-adrenoceptor agonist signaling, although such agonists can on the one hand enhance plasma membrane insertion of BTRs but also desensitize BTRs. Thus BTRs continue to be intriguing in terms of signaling in ASM.

While the pathways of [Ca²⁺]_i effects of BTRs are being teased out, the mechanisms of reduced contractility per se remain unresolved. A recent study suggests that effects on contractility may depend on TAS2R-bronchoconstrictor GPCR interactions, but the final pathways still lead to [Ca²⁺]_i first and what downstream pathways are differentially influenced by TAS2R are not known. Inhibition of Ca²⁺ sensitivity (i.e., MLCK/MLC and actin/myosin) or sensitization (RhoA/ROCK) are obvious targets, but there is currently no information on these aspects. Interestingly, caffeine, which is also a TAS2R agonist, appears to influence actin polymerization in ASM (519). Furthermore, the relative roles of the different types of TAS2Rs in the airway in the context of normal function and contribution to diseases such as asthma or COPD (relevant to the eventual utility of bitter tastants in therapy) remain to be elucidated. Here, a single recent study suggests a novel role for BTRs as an antimitogen in ASM (473). The intriguing aspect here is that bitter tastants provide antimitogenic potential without activating the recently recognized pathways of TAS2R activation, but instead inhibit growth factor activated protein kinases, and induction of transcription factors such as AP-1 and STAT3 that are important in asthma.

In line with emerging mechanisms, there continues to be new discoveries in the role GABA, the major inhibitory neurotransmitter in mammalian central nervous system. To recall, GABA acts at both ligand-gated ionotropic GABA_A receptors as well as G protein-linked metabotropic GABA_B receptors. Clinically, the GABA_B receptor agonist baclofen has been shown to worsen methacholine responses of asthmatic airways (136). In the airway, the epithelium appears to be a significant source of GABA (166). Functional GABA_B receptors in ASM (166, 167, 388) and epithelium (364) have been shown. Although acting through G_i, baclofen and GABA increase IP₃ and [Ca²⁺]_i in the airway (363), an effect involving a Gβγ-mediated direct effect on PLCβ, and potentiation of G_q-mediated signaling. Conversely, GABA_A receptors on ASM appear to be potent bronchodilators (122, 164, 165, 167, 168, 365, 581, 584). Here, one recent publication demonstrated the opportunity to selectively target GABA_A receptors utilizing the fact that only α4 and α5 subunits are present in the receptors of human ASM (168). Such emerging data point to novel and nuanced approaches to selectively targeting the heterogeneity of receptor features in the ASM to induce bronchodilation. Indeed, it may not be just GABA but also glycine receptor (a Cl⁻ channel) that is involved in bronchodilation (580), another mechanism that remains to be explored further.

It appears that GABA is not the only ligand typically associated with the central nervous system (CNS) that is also involved in ASM. The receptor for N-methyl-d-aspartate (NMDA), a glutamate analog, has been recently localized to human ASM cells (14) and is upregulated by cytokines (15). The rationale for exploring this unexpected pathway has been the association of increased plasma glutamate levels in airway inflammation, raising the question of whether glutamate receptors such as the NMDA receptor can contribute to AHR. NMDA receptor activation appears to increase [Ca²⁺]_i in ASM cells, particularly involving the NR1 subunit. However, in the context of cytokine exposure, NMDA receptor activation appears to have dichotomous effects with increased [Ca²⁺]_i in ASM per se, but bronchodilation in vivo, which may additionally involve the NOS pathway and thus neuronal or even epithelial modulation.

Finally, in the context of CNS-related ligands, dopaminergic signaling may also be relevant (75, 366, 367). “D1-like” receptors couple to G_s while “D2-like” receptors couple to G_i (361), and both types of dopamine receptors have been localized to ASM, although dopamine normally induces bronchodilation, particularly in asthmatic subjects.

An exciting and emerging GPCR is the CaSR. The CaSR is well-known for [Ca²⁺]_o regulation [via effects at the parathyroid gland, kidney, and bone (8, 181, 522)], but it is also expressed in noncalciotropic tissues such as blood vessels, breast, and pancreas, where it can regulate [Ca²⁺]_i, gene expression, ion channels, cell fate, and ECM (55, 64, 370, 434, 435). Altered CaSR expression is associated with inflammation, vascular calcification, and cancers (64, 370, 434, 435). However, beyond reports of CaSR in lung cancers (411, 552) or neuroendocrine tumors (308), there is surprisingly very little information on CaSR expression or function in the airway (111, 158, 360, 577). This paucity of information is increasingly being corrected, with recent publications on functional CaSR in the lung (65, 397, 433, 503, 518, 577) (although admittedly its role in the airway per se is still being investigated). CaSR appears to be important in developing lung although information in this aspect is minimal. CaSR is expressed in developing epithelium (158) and modulates branching morphogenesis at adult [Ca²⁺]_o levels (∼1.2 mM), while higher fetal [Ca²⁺]_o levels [∼1.7 mM (285)] may inhibit branching. What CaSR does in postnatal airways is unknown, although emerging data on its role in regulating human fetal lung development via the CFTR (65) is intriguing. In adults, CaSR is present in human airways (577), particularly in ASM, where it regulates [Ca²⁺]_i and contractility (577). Furthermore, CaSR expression is increased in ASM of asthmatic subjects (577), making it a potential target for mechanistic exploration as well as a future therapeutic avenue. Here, the importance of CaSR may lie in the multiple endogenous modulators beyond [Ca²⁺]_o, with CaSR responding to polyvalent cations (particularly spermine and eosinophil cationic protein), amino acids (e.g., l-arginine), ionic strength, and alkaline pH (434, 435) (Fig. 4). Depending on cell type and context, signaling pathways such as RhoA/ROCK, ERKs, PKC, and cAMP/PKA can all regulate CaSR expression and function (434, 435), making this receptor a multimodal sensor and effector for integrating multiple signals, many of which happen to be important in airway structure and function (Fig. 4). In this regard, emerging data on a pathophysiological role for CaSR in pulmonary artery smooth muscle (198, 400, 518, 566, 567) highlights the potential for this pathway in the airway as well: an area of unmet need. Here, the arginases may be particularly relevant, given that their enzymatic activity leads to shunting of l-arginine toward the ornithine pathway leading to production of the polycation spermine. Indeed, there is increasing interest in the role of arginases and polyamines in asthma (5, 61, 104, 172, 331, 467, 474) and even pulmonary hypertension (89, 95, 194, 254, 393, 566) (also see discussion on arginases further below). Certainly, other aspects of CaSR regulation (as above) remain to be explored in any cell type of the lung. Given the recent discovery of CaSR in asthmatic ASM, this not-so-novel GPCR may see an emergence in the context of lung disease.

Fig. 4.

Calcium sensing receptor (CaSR), a novel signaling mechanism in ASM known for its activity in the parathyroid gland. Recent data suggest the involvement of CaSR in elevating [Ca²⁺]_i and contractility in human ASM, especially in the context of inflammation which increases CaSR expression. Normally, CaSR is sensitive to extracellular Ca²⁺ ([Ca²⁺]_o) and is modulated by polyamines (e.g., spermine, eosinophilic cationic protein), pH, and amino acids such as l-arginine. Intracellularly, CaSR activates the PLC/IP₃ pathway, but also enhances PKC, RhoA/ROCK, ERK, and other cascades that can contribute to increased [Ca²⁺]_i and proliferation. Conversely, CaSR can block cAMP and thus impair bronchodilation. The potential importance of CaSR lies in the existence and trialing for CaSR agonists (calcimimetics) vs. antagonists (calcilytics) for other diseases.

In the context of continued discoveries of GPCRs in bronchoconstriction vs. dilation, the complex roles of prostaglandins (PGs) in the airway (99, 215, 372, 447, 455, 586) are being teased out. Products of the COX pathway in arachidonic acid metabolism, the five primary PGs (PGD₂, PGE₃, PGF₂, PGI₂, and TXA₂), all signal through distinct GPCRs. Complexity arises from the fact that some PGs counteract actions of others, or that the same PG may have opposing effects depending on the specific receptor involved, which is further context sensitive. Here, thromboxane (TXA₂) is one of the most potent endogenous bronchoconstrictors across several species, exerting biological functions through interactions with the thromboxane-prostanoid (TP) receptor coupled to G_q, and thus the PLC/IP₃/Ca²⁺ pathway (Fig. 5). Increased TXA₂ levels are seen in asthmatic airways (555) and in COPD (125), while inhibition of TP receptors attenuates allergic bronchoconstriction while TP inhibition attenuates bronchoconstriction. Here, TP expression on ASM has been demonstrated (11, 79, 245), supporting the idea of direct action on ASM to induce bronchoconstriction. However, TXA₂ effects appear more complex, involving indirect actions via neuronal stimulation leading to ACh release as well as mechanical stimuli (120, 121, 215, 247, 456). Furthermore, the relative role of ASM TP vs. neuronal TP appears to depend on the state of allergic inflammation, with ASM TP being more active under normal conditions (114), thus raising the question of whether targeting TP on ASM for bronchodilation will be sufficient. Nonetheless, TP may play other roles in smooth muscle in terms of actin polymerization (and thus tissue stiffening) (156, 157). Furthermore, interactions of TP with other GPCRs could be relevant to understanding whether specific constricting (263, 310) or dilating agents (420, 476, 510) (such as BTRs) may be more potent under certain conditions.

Fig. 5.

Eicosanoids in ASM. Both established and emerging data indicate a role for the thromboxane (TXA₂) receptor (TP) to increase [Ca²⁺]_i and enhance actin polymerization in ASM, leading to bronchoconstriction and airway stiffening. Prostaglandin E2 (PGE2) can activate 1 of 4 epoprostanoid receptors (EP1–EP4) with differing effects, where EP2 and EP4 reduce [Ca²⁺]_i by activating cAMP, while EP1 serves to increase [Ca²⁺]_i and EP3 to promote mitogenic activity.

In addition to TXA₂, there is resurging interest in the potential beneficial effect of PGE2 and the complex role of epoprostanoid (EP) receptor subtypes in bronchoconstriction vs. dilation. The relevance of PGE2 goes beyond direct effects on the airway, with influences on responsiveness of the lung to infection (139, 195, 238), immune cell function (140, 569), epithelial and endothelial function (24, 186, 278, 316, 356), and even senescence (207). PGE2 is abundantly produced by both airway epithelium and ASM (97, 130, 569). Previous studies suggest endogenous PGE2 is bronchoprotective in human asthma (395). Epithelially derived PGE2 reduces vagal cholinergic contraction of ASM (38) with additional effects on the immune system (464). Conversely, such epithelial PGE2 can induce ASM to produce amphiregulin (for example) to feedback onto epithelial EGFR (127). PGE2 can inhibit ASM migration and proliferation when used in combination with β₂-adrenergic receptor agonists (182, 568). Furthermore, lack of the microsomal PGE2 generating enzyme mPGEs1 results in defective clearance of bacteria (139). Accordingly, there should be substantial interest in PGE2 agonism. PGE2 signals through four distinct GPCRs, EP1-4 with each EP receptor having distinct G protein coupling and downstream signal, with complexity arising from the fact that these downstream pathways can be counteractive (99). EP1 increases Ca²⁺ while EP2 and EP4 increase cAMP to induce bronchodilation (Fig. 5). While this dichotomy should facilitate interest in inhibitors of EP1 with concurrent promotion of EP2 or EP4 activation to enhance bronchodilation in asthma (25, 45, 451), previous trials may not have been as successful due to the fact that tested drugs with broad EP agonism can also activate EP3, which causes ASM contraction by decreasing cAMP synthesis. Furthermore, EP3 activation mediates the cough induced by PGE2 (334). Such differential, counteractive roles of EP3 are also noted in the context of ASM migration (25). Accordingly, there is now interest in exploring EP2/4-specific agonism to limit the side-effect profile of PGE2. However, even here, it may be important to consider species differences in EP receptor subtype express and function that makes interpretation complex. For example, a recent study found that a traditional Chinese medicine herbal formula ASHMI dilates ACh-induced mouse airways via EP2/4 signaling (498), while another study found that in human bronchial rings it may be EP4 that is particularly significant for bronchodilation (45). This may be important in the context of recent findings of anti-inflammatory effects of EP4 (51).

There continues to be development in our understanding of novel roles for Wnt signaling in the airway, including in ASM (26, 248, 291, 292, 295, 431, 486, 495, 541, 579, 600), epithelium (175, 211, 291, 486, 496, 544, 549, 565, 588, 600), and fibroblasts (357, 376, 495, 535). Wnt proteins bind the extracellular domain of receptors of the Frizzled (Frz) family of GPCRs (332, 431), facilitated by coreceptors such as lipoprotein receptor-related protein (LRP)-5/6, Ryk, and ROR2. Wnt signaling can integrate inputs via canonical β-catenin-dependent and -independent pathways and noncanonical Wnt/Ca²⁺ pathways. The emerging relevance of Wnt signaling in ASM appears to be in remodeling, particularly in the context of TGF-β interactions and ECM production (291, 292, 431). However, TGF-β-independent effects may also be involved, as recently suggested by reports of eosinophil-induced enhancement of ASM Wnt/β-catenin expression and signaling (248). In fibroblasts, Wnt5B can increase IL-6 and CXCL8 secretion and thus indirectly influence airway remodeling (535). Furthermore, remodeling can be indirectly mediated via Wnt signaling in epithelial cells that produce ECM in the context of inflammation (588, 600). Noncanonical Wnt signaling (e.g., via Wnt5a) can involve effects on [Ca²⁺]_i (292, 579). Thus Wnt signaling has the potential to substantially influence airway structure and function.

Non-GPCR mechanisms.

A large number of non-GPCR pathways have been explored in the context of airway contractility and relaxation and cannot be reasonably summarized here. Nonetheless, some specific pathways have gained recent interest.

Relevant to airway irritability, and the potential for airway innervation to influence contractility, the role of transient receptor channels (TRPs) has been explored, with particular emphasis on the TRPA1 and TRPV1 channels (88, 271, 315, 334), that are activated by PKC [e.g., following stimulation of GPCRs such as bradykinin or prostanoid receptors (334)]. However, there is now increasing evidence that ASM also expresses TRPA1 (88, 251, 374, 590), TRPV1 (362, 551, 593, 594), and, similar to the vasculature (31, 115, 371, 425, 539, 572), TRPV4 (252, 350, 511, 592). Here, TRPA1 has been shown to enhance ASM-derived IL-8 secretion (374), to enhance airway inflammation and AHR (374), as well as to mobilize [Ca²⁺]_i (251) (Fig. 2), while conversely inhibiting ASM proliferation (590). In contrast, TRPV1 on ASM appears to promote proliferation (593, 594). However, TRPV4 appears to be involved in elevation of [Ca²⁺]_i (592) and contractility (350) as well as release of procontractile factors (350, 511). The relevance of these emerging pathways lies in leveraging previous neuroscience work on TRPA and TRPV channels for benefit in airway disease. Further relevance lies in linking such TRP channels to other mechanisms for airway irritability, such as acid-sensing ion channels that are found in ASM as well as vascular smooth muscle (152, 185, 412), and the emerging family of proton-sensing GPCRs (e.g., OGR1 or GPR68) that respond to extracellular pH (237, 347, 384, 462, 524).

Although well recognized in the context of epithelial dysfunction in cystic fibrosis (106, 422), the CFTR appears to now be expressed in ASM and is functional and potentially contributory in airway disease (1, 65, 110, 352, 359, 543). While its expression profile and functionality are still under exploration, it appears that ASM CFTR contributes to SR Ca²⁺ function (110, 359), and, importantly, altered CFTR function can lead to AHR (7, 352, 543). Conversely, administrator of CFTR-targeting drugs appear to promote bronchodilation (1, 379). These exciting findings suggest an entirely novel area for investigation in the context of ASM mechanisms that contribute to AHR.

An interesting aspect of ASM signaling is the role of EGFR (305, 317, 375, 386, 463, 482, 570), typically associated with epithelial cells (62, 127, 193, 261, 597). Emerging data suggest that in addition to secreting amphiregulin (EGFR ligand) (127), ASM also express EGFR, which can be involved in signaling of procontractile agonists such as endothelin-1 (ET1) (317) and brain natriuretic peptide (BNP) (386) as well as airway inflammation (482).

An interesting pathway in the context of Ca²⁺ regulation that has been recently reported is calcineurin/NFAT (185, 201, 461, 491, 592, 595). Although calcineurin can mediate its effects via the transcription factor NFAT, a recent study reported that calcineurin can also modulate local Ca²⁺ signaling and contractility in ASM, specifically via RyR channels (461) (Fig. 2). Conversely, local Ca²⁺ signals created by TRPV4 can activate calcineurin to further increase ASM Ca²⁺ and proliferation (592). Furthermore, the Ca²⁺ influx channel TRPC3 can activate the calcineurin/NFAT pathway in modulating airway contractility (491). Beyond Ca²⁺, calcineurin also appears to be involved in modulation of actin polymerization and tension development in ASM (595). The relevance of calcineurin lies in the well-known importance of its inhibition in immunosuppressive therapies, and their potential application in airway disease.

Unexpected mechanisms modulating Ca²⁺ and contractility are increasingly being recognized. For example, there is much interest in the role of the glycosaminoglycan hyaluronan, a major component of the ECM, in modulating a number of aspects of airway disease (149, 170, 265, 279, 298, 301–304, 336, 391, 492, 502, 559). It appears that ASM itself produces hyaluronan, and increased expression occurs in the context of inflammation while steroids can reduce such expression (279, 301, 391), raising the question of what role(s) hyaluronan could play in the airway. Hyaluronan is involved in water homeostasis, cell-matrix signaling, cell proliferation and migration, as well as modulation of inflammation (170). Here, emerging data suggest that high- vs. low-molecular-weight hyaluronan may play differential roles in enhancing vs. alleviating disease processes (304). Exogenous hyaluronan can prevent inflammation and, importantly, blunt AHR, including reducing ASM Ca²⁺ and contractility in the context of injury (170, 304), thus making it an exciting and novel potential therapy.

Novel ASM Mechanisms in Airway Remodeling

Diseases such as asthma and COPD are well appreciated to involve extensive airway remodeling, although what constitutes remodeling per se is defined loosely or at least differently in different studies. Remodeling was recognized quite early as airway wall thickening and mucus plugging in severe asthma (233), with more recent studies better delineating, characterizing and quantifying structural changes with remodeling (57, 74, 82, 94, 147, 150, 170, 255, 277, 295, 390, 392, 414, 417, 431, 446, 471, 473, 479, 482, 529, 540, 556). Changes include thickened epithelium with mucus gland hypertrophy (likely contributing to the increased mucus plugging), subepithelial membrane thickening and fibrosis with altered ECM composition and deposition, and increased ASM mass reflected by both hyperplasia and hypertrophy (35, 42–44, 129, 146, 240, 431, 473, 540). An important aspect of remodeling is the substantial heterogeneity in the type and extent of changes that occur in airways of different sizes with large bronchi showing cellular hyperplasia for example, while other parts of the airway showing hypertrophy (42, 146). Furthermore, the models and systems used to explore remodeling differ with the use of in vitro models of animal or human ASM (or other cell types), and preclinical in vivo models (71, 220, 281, 290, 351, 500). While these models have certainly enhanced our understanding of remodeling and the role of ASM for example, the longitudinal and progressive aspects of remodeling continue to be limiting for our understanding of the underlying mechanisms, particularly at different stages of airway disease. Conversely, explorations using such models may help us understand whether aspects of AHR not responsiveness to current therapy represent remodeling (107, 173, 188, 293, 394, 396, 438, 441, 446, 562).

In terms of ASM hypertrophy, both mechanical and inflammatory pathways could be contributory. Mechanical stretch induces ASM hypertrophy via Wnt and GSK3β (295), Akt (43, 330, 369), and mTOR (598) [especially when stimulated by TGFβ or endothelin (180, 355, 596)]. Other hypertrophy mechanisms have also recently been reported such as SRF and Elk1 (94) (Fig. 6); these have not been examined in ASM per se, although substantial insight could be obtained from emerging data in pulmonary vasculature for signaling intermediates and transcription factors such as the CaSR (198, 397, 566), G6PD (93), Klf5 (312), and Akt1 (517).

Fig. 6.

Role of ASM in remodeling. In addition to an essential role in contractility and bronchodilation, ASM acts as recipient of extrinsic factors such as allergens, infection, environmental factors, cytokines, growth factors, and even the extracellular ASM. In turn, ASM demonstrates “synthetic” aspects, itself producing a range of ECM proteins, matrix metalloproteinases (MMPs) and inhibitors (TIMPs), pro- and anti-inflammatory cytokines, growth factors such as neurotrophins and VEGF, and a range of other proteins. The relevance of these activities lies in the hypertrophy vs. hyperplasia of ASM as well as fibrosis that contribute to airway remodeling in disease. Here, the factors (extrinsic or ASM-derived) that contribute to hypertrophy vs. hyperplasia can differ and could include mechanical forces, signaling cascades and emerging mechanisms such as miRNAs, mitochondria, endoplasmic reticulum (ER) stress, etc. Furthermore, some mechanisms can blunt hypertrophy and hyperplasia. See text for various abbreviations in figure.

A number of mechanisms underlying ASM hyperplasia have been reported (Fig. 6) and involve upregulation of cell proliferation (or inhibition of processes that prevent it), or alternatively inhibition of apoptosis. As with hypertrophy, mechanical stretch alone can induce proliferation, working through Wnt or altered matrix stiffness (478). Mechanical stress in airways can also induce EGF release from epithelial cells and indirectly contribute to remodeling (477). Beyond stress, a range of inflammatory stimuli are known to enhance ASM proliferation, including the classic Th1 and Th2 cytokines such as TNF-α, IL-4, IL-5, and IL-13, as well as TGFβ, TSLP, IL-6, and those of the Th17 family (35, 85, 129, 131, 138, 236, 270, 342, 407, 428, 429, 439, 536). Such stimuli activate “classic” pathways such as p38 and p42/44 MAP kinases, PI3/Akt, and NF-κB (18, 85, 117, 190, 401, 404, 439, 473, 491, 594).

In addition to inflammatory stimuli, several new triggers for proliferation have been noted. Even influx channels could be involved, as exemplified by TRPC1 (491) and TRPV1 (594), while, conversely, activation of TRPM8 and TRPA1 channels appear to inhibit proliferation (590). Regardless of their mechanosensitive role, Wnts can modulate ASM proliferation (248, 431, 486). Locally produced factors such as VEGF (338), neurotrophins (18, 128, 163), amphiregulin (305, 481), prostaglandins (536), and leukotrienes (353, 508, 515) could be involved. And finally some studies have reported a higher proliferation of asthmatic ASM cells at baseline (257, 530), although the underlying mechanisms are not completely understood. However, it should also be emphasized baseline higher ASM proliferation in asthmatic subjects is not conclusively proven (266, 550). Thus a plethora of mechanisms are potentially involved in increasing ASM mass in the context of remodeling, with their relative contribution likely depending on context.

What are less understood are the potential mechanisms that limit ongoing proliferation, especially in the context of inflammation (which also needs limitation). Therapeutic agents such as corticosteroids and β₂-agonists can limit proliferation (48, 182). Recent studies have also identified pathways that can limit smooth muscle proliferation including the structural protein caveolin-1 (22, 189, 190), the channels TRPM8 and TRPA1 (590), bitter taste ligands (473), PPARγ ligands (141, 142, 160, 161, 177, 321, 432, 501, 511, 560), and vitamin D (102, 118, 329, 339, 583). Although the above discussion, albeit brief, highlights some pathways for limiting remodeling, there is surprisingly little information regarding how inflammation effects are suppressed under normal conditions. Obviously, drugs such as glucocorticoids can suppress NF-κB and other pathways, as well as activating anti-inflammatory cascades. However, in other lung cell types, including pulmonary artery smooth muscle, there is increasing evidence for the resolvins (particularly D1) (112, 219, 253, 387, 419, 476), reducing contractility, cell migration, and other features that are common to airway remodeling. Conversely, in the context of responsiveness to infectious and other stimuli mediated by Toll-like receptors (TLRs), activation of the inflammasome is thought to be important for homeostasis. The inflammasome is an intracellular multimeric protein complex regulating maturation and release of proinflammatory cytokines such as IL-1β and IL-8 in response to pathogens and endogenous danger signals. There is now emerging evidence that the inflammasome itself plays a role in respiratory diseases. However, there is currently little information regarding factors that activate (or not activate) the inflammasome in ASM (221, 311, 416, 489). Understanding the role of factors such as resolvins and the inflammasome represent novel areas for research in the context of ASM and airway disease.

In addition to fibroblasts, ASM is now also well recognized to generate a range of ECM proteins (35, 47, 52, 73, 128, 222, 291, 450, 562), which has bearing given that the increased fractional area of the matrix within the smooth muscle layer of fatal asthmatics (29). Furthermore the ECM network can passively and actively modulate cell proliferation and migration, and thus influence airway structure and function. Here, ASM cells of asthmatic subjects may not only produce more ECM, but also a different profile. The ECM proteins of particular relevance in the context of ASM include collagens, fibronectin, versican, tenascin, perlecan, decorin (59, 72, 226, 241, 256, 295, 502), and members of the MMP family (MMP-1, -2, -9, and -12) (98, 267, 286, 296, 382, 507) [although some proteins such as collagen IV and elastin decrease (72)]. Furthermore, there is increasing interest in the transmembrane integrins (particularly α5 and β1) that can mediate and modulate cell-cell and cell-ECM interactions (562). Here, the ECM can also regulate activity of local growth factors (e.g., neurotrophins, VEGF) and cytokines by cleavage and inactivation (148, 176), creating a complex network to regulate remodeling. Accordingly, altered ECM production, e.g., modulated by inflammatory mediators or growth factors produced either by ASM itself or other cells, can have interactive effects on airway structure and function. This has further significance in the context of whether and how therapies such as glucocorticoids can influence airway structure (i.e., remodeling) by modulating the local inflammatory environment, and thus in turn the ECM.

A number of signaling pathways in the context of inflammation and ECM have been explored and some are highlighted here (72, 286, 457, 532, 536). Here, ASM can produce a wide variety of pro- and anti-inflammatory factors (37, 225, 230, 286, 427, 428, 484, 488, 512), including IL-1β, IL-5, IL-6, IL-8, IL-17, PDGF, TNF-α, TLSP, and TGFβ, although their autocrine/paracrine function in the context of remodeling are still being explored. For example, IL-6 can induce ASM hyperplasia and further modulate immune cell function. TNF-α can mediate effects by enhancing IFNβ secretion (346) while TGFβ can regulate IL-6 release (358). There are several examples of such cytokines influencing ECM and remodeling. For example, IL-1β can interact with TNF-α to increases MMP-12 (564) and MMP-9 (313), which can promote cell migration and remodeling and can further modulate growth factor activity (148, 176). TGFβ enhances deposition of perlecan (235), while Wnt/β-catenin pathway can regulate TGFβ effects on ASM-derived ECM (27, 286). Conversely, decorin, an ECM proteoglycan, binds TGFβ and blunts ECM production (343). Downstream, glucocorticoids can inhibit TNF-α-induced ECM production by activating the NF-κB inhibitor TNAIP3 (457). These examples highlight ASM involvement in ECM and remodeling.

ASM can also produce chemokines such as RANTES, eotaxin, MCP-1, CXCL10 (119), CXCL8 (103, 127, 153, 197), and CXCR3 ligand (516), although their effects in the context of remodeling are likely indirect via recruitment of inflammatory cells. In addition, ASM is now increasingly shown to produce growth factors such as BDNF (20, 418) and VEGF (127, 439, 478) that can also affect remodeling by enhancing ECM production.

Beyond inflammatory mediators and growth factors, a number of emerging mechanisms have been reported in the context of ASM and airway remodeling. One example is vitamin D, which has been shown to suppress remodeling both in vitro and in vivo (33, 66, 67, 102, 118, 268). This is an intriguing concept given recent reports that calcitriol is not additionally beneficial in severe asthma (63, 83). However, it also important to note that the mechanisms by which vitamin D influences airway cells are still under investigation, with recent studies attempting to explore differential gene regulation by this vitamin (162). Surprisingly, other vitamins in the context of remodeling have barely been examined with a single report on vitamin E (205). Another emerging mechanism is thyroxine, which appears to enhance ASM proliferation (129) and in the context of airway development results in malformed airways when thyroxine levels are low (179). Similarly, insulin appears to enhance ASM proliferation and ECM formation (5, 485) and may be important in the context of metabolic syndrome and other conditions of elevated insulin levels (6, 486, 487) as well as lung development (349, 385). Another emerging topic is the role of sphingolipids in airway inflammation, AHR and remodeling particularly the effect of sphingosine-1-phosphate in promoting contractility, inflammation and remodeling via ASM (86, 87, 440, 442, 448, 449, 534). Finally, the potential role of transglutaminase has also been preliminarily examined (203) although it has been studied more in the context of allergy, asthma and epithelial function (381, 383, 494) and in pulmonary hypertension (399, 400). The relevance of these disparate mechanisms lies in understanding the complex inputs and outputs with ASM at the center in the context of remodeling, only making it more clear that remodeling will not be easy to target.

Epigenetic Mechanisms and ASM

There is now increasing interest in and evidence for epigenetic regulation of multiple aspects of ASM structure and function in the context of disease. Epigenetics involves altered patterns of gene expression resulting from chromosomal changes that do not involve alterations in the DNA sequence. Epigenetic changes alter accessibility of promoters and enhancer/silencer regions within chromatin to transcription factors and thus regulate global activation or local transcription for specific genes. Such changes are mediated by a number of mechanisms such as posttranscriptional modification of histone proteins [acetylation and deacetylation via histone acetyltransferases (HATs) and deacetylases (HDACs)], miRNAs and noncoding RNAs (ncRNAs), and DNA methylation. The combination of these many mechanisms results in complex, potentially interactive effects on gene expression. Accordingly, even with increasing data on epigenetic mechanisms in ASM in the context disease, it becomes difficult to assess whether inhibition or activation of specific epigenetic pathways will alleviate vs. worsen disease. Nonetheless, there is now substantial interest in therapeutic avenues such as targeting of miRNAs, HDAC inhibitors, etc., for asthma and COPD. However, it is important to stress that the evidence to date is not necessarily convergent at this stage.

Initial evidence for abnormal histone modifications in asthmatic ASM comes from limited studies showing that enhanced CXCL8/IL8 (103) and VEGF (101) secretion involve aberrant HAT binding and resultant histone H3 acetylation at their respective promoters. Conversely, trichostatin A (a class I and II HDAC inhibitor) blunts the inhibitory effect of IFNγ on TNF-α-induced CXCL8 secretion in human ASM (269). Along similar lines, TNF-α induces H4 acetylation of the eotaxin promoter (269, 469), while TNF-α and IFNγ induce H4 but not H3 acetylation at the CXCL10 promoter (100). These disparate pieces of evidence highlight the potential for histone-based epigenetic mechanisms in immunomodulatory roles for ASM.

In the context of ASM function, there are only limited data for histone modification. For example, HDACs are downregulated in diseases such as asthma and COPD (36, 444–446) and may contribute to corticosteroid insensitivity. Accordingly, histone modifications could influence contractility, remodeling as well as inflammation, and further make HDAC upregulation (not downregulation) a novel therapeutic strategy. Yet it is the nonselective HDAC inhibitor trichostatin A that has been found to blunt ASM contraction (34), while other studies show that trichostatin A can blunt airway inflammation (229, 443, 523). This only highlights the complexity of these epigenetic mechanisms in the airway.

In terms of ASM cell proliferation as an aspect of remodeling, there is little to no evidence for epigenetic regulation. A single study found that specific bromodomains that control histone acetylation could be altered in asthmatic ASM and influence proliferation (409). However, there is increasing evidence for histone acetylation in pulmonary artery smooth muscle proliferation (380, 571) that may provide insight into potential roles in ASM. Similarly, there is no information on epigenetic regulation of ASM-derived ECM formation, but again emerging data from the vasculature (84, 183, 306, 413, 554) may provide insight.

Compared with the limited data on histone modifications, there are considerably more studies on miRNA regulation of ASM. As with most cell types or conditions where miRNAs are explored, a multitude of candidate miRNAs have been identified as playing multiple roles in ASM, making it again difficult to evaluate their relative contributions to normal ASM structure/function vs. disease. Furthermore, given that the same miRNA may or may not have the same targets in ASM of different species, data correlation between in vitro work (typically done in human ASM) to in vivo studies in mouse models of asthma, or where specific miRNAs have been up/downregulated, becomes difficult. One initial study profiled miRNAs in airway biopsies of subjects with mild asthma and surprisingly found no major or consistent changes in miRNAs to suggest a role in this condition, or in the responsiveness to steroid therapy (558). However, a number of later studies have found several miRNAs regulating specific aspects of ASM, with some studies pointing to particular miRNAs with broader function (interpreted as having broader and perhaps substantial role in disease pathophysiology). For example, one study in human ASM showed that exposure to proinflammatory cytokines such as IL-1β, TNF-α, and IFNγ downregulated 11 miRNAs, particularly miR-25, miR-140, miR-188, and miR-320, with miR-25 having a broad role in modulating expression of a range of inflammatory mediators, ECM, and contractile proteins and the transcription factor KLF4 (289). While this study showed the typical downregulation of multiple “protective” miRNAs, another study (299) showed that miR-146, which is widely induced in inflammation, is also enhanced in ASM with IL-1β exposure but does not mediate IL-1β-induced cytokine release by ASM. On the other hand, another group also explored miR-146a and miR-146b in human ASM from asthmatic and nonasthmatic subjects treated with cytomix (IL-1β, TNF-α, IFNγ) found elevated miR-146a and miR-146b, particularly in asthmatic patients. However, only miR-146a (which negatively regulates COX-2 and IL-1β) was functionally important (108). These contrasting results highlight the difficulty of simply correlating miRNA expression with specific functions. Another study showed that miR-140-3p regulates the important enzyme CD38 (259), which could have a multitude of downstream effects including [Ca²⁺]_i and proliferation (133, 138, 196). Interestingly, mechanical stretch induces miR-26a that indirectly acts to induce ASM hypertrophy by blunting GSK3β (368, 369). Yet ASM proliferation appears to be driven by multiple miRNAs, including miR-10a (232), miR-23b (91), miR-138 (325), miR-145 (324), miR-203 (314), miR-221 (408), and miR-708 (138). There is relatively less known regarding ECM modulation, with studies showing a role for miR-25 (289) and miR-145 (324) at least. While these emerging studies show the potential for multiple miRNAs in ASM itself, it would be relevant to compare and contrast similar emerging data from other cell types and conditions in the lung toward understanding the complex roles of miRNAs in airway disease. For example, in fibroblasts, let-7d (234), miR-96 (377), miR-146a (108, 354), and miR-210 (53) appear to be important in the context of fibrosis, whereas in alveoli and lung injury miR-21 (537) miR-29b (140), miR-153, and miR-155 (547, 574) and in developing lung miR-124 (549) and miR-489 (385) have been shown to be relevant. Overall, these many pieces of data point to the need for understanding miRNA regulation in ASM (and other cell types as well), but important questions remain to be addressed in this context: 1) how do conditions contributing to disease, including inflammatory mediators, growth factors, etc., influence miRNA profiles; 2) does disease severity matter; 3) how do miRNAs interact with each other; 4) what are the feedback effects of specific factors in regulating miRNAs per se; and, 5) finally, how should the in vitro data be verified in vivo, and are animal models appropriate? Even in the context of therapy, while targeting of miRNAs is now being attempted, specificity and access to particular cell types remain difficult to address, especially if miRNA function differs between cell types.

Mitochondria and ASM

There is now substantial evidence that mitochondria play substantial roles in the airway beyond production of ATP, including Ca²⁺ buffering (21, 109, 436) (which also helps regulate mitochondrial energy production to match demand), ER pathways, Ca²⁺ influx such as SOCE, cell proliferation, and survival. While these concepts have been recently studied to a greater extent in other cell types (particularly cancers), there is now emerging evidence in ASM as well (16, 17, 19, 21, 92, 116, 131, 132, 155, 178, 206, 216, 294, 344, 358, 509, 530, 557). Here, it appears that mitochondrial structure in the context of fission and fusion, mitochondrial biogenesis and mitochondrial destruction via autophagy (mitophagy), and reactive oxygen species (ROS) are all important aspects of the noncanonical contributions for mitochondria (21, 178). For example, depletion of mitochondrial DNA blunts [Ca²⁺]_i responses in ASM (92). TGFβ enhances ASM mitochondrial ROS in promoting cytokine secretion (358). Such ROS are also involved in cigarette smoke effects on ASM mitochondria (19). Conversely, inflammation impairs mitochondrial Ca²⁺ buffering that leads to elevated cytosolic Ca²⁺ (131, 132). Such impairment can lead not only to elevated ROS, but also to elevated ER stress and the unfolded protein response (UPR) (131). While the role of both ER stress and UPR in ASM per se has been minimally investigated (131, 174, 264, 389, 599), the relevance of these pathways lies in their potential to influence protein expression and function in the context of contractility and remodeling in asthma: an emerging topic of interest (78, 154, 199, 227, 231, 274, 275, 307, 337, 480, 545). Indeed, recent studies have explored the possibility of chemical chaperones that influence the UPR in the context of allergic asthma (199, 227, 337), while other studies have found links between the ER protein ORMDL3 and the UPR (231, 545).

Insight into the relevance of mitochondria in airway disease also comes from emerging data in other lung cell types and conditions where mitochondrial dysfunction appears to play a role. For example, impaired mitochondrial biogenesis contributes to pulmonary hypertension (2, 341, 465, 578), while a range of insults including cigarette smoke, hyperoxia, infection, excessive ventilation, etc., result in impaired mitochondrial function and/or ER stress in alveolar cells (4, 12, 32, 60, 90, 123, 124, 126, 187, 208, 210, 288, 326–328, 415, 533). Mitophagy also appears to be play a role in pulmonary vascular disease (3, 209, 421). Conversely, protective mechanisms, particularly the sirtuins, may be important to consider but have barely been explored in the lung (2, 169, 264, 280, 341, 520, 573). Mitochondrial dysfunction may also be relevant in lung manifestations of metabolic diseases such as diabetes (5, 6, 486) and in aging (204, 553) and senescence (204, 258, 426): topics that have been barely explored. Thus the potential links between mitochondria, ER stress, and ASM structure/function represent a novel area of research in airway disease.

Sex Differences and Sex Steroids

With the NIH's ongoing emphasis on exploring intrinsic sex differences in all diseases, it is all the more imperative to understand concepts of sex differences and sex steroid signaling in lung diseases, given clinical evidence for peripubertal and perimenopausal changes in the incidence of asthma and increased asthma in elderly men (54, 184, 460, 468, 526, 538, 587). Here, studies have explored both intrinsic and extrinsic (steroid-mediated) effects (137, 297, 345, 452, 499, 513, 527, 528, 546). What makes this topic intriguing, yet difficult, is separating intrinsic differences between men and women (or boys vs. girls) in airway structure/function, particularly in disease, from the extrinsic effects of sex steroids (56, 348, 460, 528). Here, it could be argued that intrinsic differences are what they are, and while it is important to understand such differences in the context of age-dependent changes in the airway, the superimposed effects of sex steroids from fetal stages (both fetal and maternal steroids) onward through puberty and beyond make it relatively more important to understand the extrinsic effects of sex steroids. In this regard, estrogens, progesterone, and/or testosterone can be expected to work both genomically and nongenomically, but it is not clear that any or all of these sex steroid effects are consistent across cell types or even within a specific sex. Akin to their role in vascular smooth muscle, estrogens can rapidly (nongenomically) reduce [Ca²⁺]_i in ASM, working via a variety of pathways (528), acting via ERα and ERβ (527). Whether such estrogen effects are maintained in asthma is not known, although unpublished data from our groups (Sathish et al., unpublished observations) suggest that inflammation and/or asthma lead to an altered balance of ERα vs. ERβ levels, with a greater influence of ERβ. In the context of remodeling, there are both intrinsic sex differences (513) (although the role of ASM per se is not known), as well as different effects of estrogens vs. testosterone (137, 499). Furthermore, ER variants appear to also be important in female asthmatic subjects (137). Whether such variants are also important in men, or whether ER profiles differ between the sexes, with age and pubertal status, etc., are all unknown. It is also important to consider whether and how progesterone influences ASM with limited data of effects on the airway function (200, 213, 239, 514). A similar question can be raised regarding whether testosterone, acting via androgen receptors, is bronchodilatory or protective as suggested by some studies (77, 80, 81, 283, 284, 410) and whether differences in receptor regulation between men vs. women play a role (284, 585). Overall, these many unanswered questions represent another important area for investigation in airway disease, particularly the role of ASM.

ASM in Airway Development

There is increasing evidence that factors early in airway development can influence not only normal lung growth, but also airway diseases throughout life such as such as asthma or COPD (39, 144, 475). While postnatal airway growth and its dysfunction can be studied in animal models as well as in babies and children, even if not as easily as in adults, our insight into processes regulating prenatal airway development has also been facilitated with approaches such as lineage studies, transgenic animals, and imaging techniques. Here, understanding bronchial airway growth may be particularly important given that the number of airways is fixed at birth, unlike continued postnatal development of alveoli.

The ASM likely plays a role in modulating developing airway structure and function. Even if the prenatal airway is not involved in ventilation per se, airway peristalsis has been shown to occur in vitro (249, 250) with cyclical Ca²⁺ waves within fetal ASM cells. Such processes may promote airway branching and elongation and underline a functional role for ASM. What mechanisms underlie airway peristalsis is not clear. In adult ASM (and in other smooth muscle cell types), baseline [Ca²⁺]_i oscillations modulated by agonist stimulation have now been shown extensively (70, 223, 406, 425, 459, 521). Many of the [Ca²⁺]_i regulatory pathways involved in adult ASM are also present in developing ASM (206) and could play a role. An intriguing additional mechanism may be the CaSR. Although CaSR is known to be expressed in developing epithelium (158) and modulates branching morphogenesis at adult [Ca²⁺]_o levels (∼1.2 mM), the higher fetal [Ca²⁺]_o levels [∼1.7 mM (285)] cause CaSR to inhibit branching. However, it is possible that CaSR is also expressed in fetal ASM and given the higher [Ca²⁺]_o is more sensitive and thus elevates ASM [Ca²⁺]_i, setting the stage of [Ca²⁺]_i oscillations and peristalsis. Alternatively, oscillations in membrane potential and cyclical Ca²⁺ influx (again facilitated by higher [Ca²⁺]_o) may be involved. The relevance of understanding these pathways lies in determining causes of lung hypoplasia, and particularly the effects of iatrogenic insults such as hyperoxia, mechanical stretch of CPAP, and mechanical ventilation in the context of premature birth and respiratory support of the immature neonate (217, 218). Here, the emerging role of mechanosensitive pathways in modulating expression of genes and proteins should be a model for understanding how mechanical forces within the developing airway induced by ASM cells as well as external forces on ASM influence airway growth. These processes could be modulated by growth factors and transcription factors in the context of normal lung development, such as FGF10 (191, 250, 466), neurotrophins (171, 423, 493, 575, 576), and Wnt (202).

Beyond Ca²⁺ and growth factors, an intriguing aspect of airway development in the context of subsequent disease in childhood or adults is epigenetic effects that could occur in utero to influence asthma (58, 144, 214, 228, 273) and even traverse generations, particularly in cigarette smoke effects, and contribute to COPD (287, 309, 506). Whether ASM per se is involved is not known but again makes for an exciting area for investigating its role in identifying developmental origins of adult disease.

Emerging Topics in ASM Biology

There are understandably a number of areas where the role of ASM is being or should be explored but cannot be justifiably summarized here. Indeed, one of the limitations of introducing or discussing these topics is that they have been addressed either in barely a few studies, done entirely in vitro (sometimes in cells from nonhuman species), in a very limited context, or are hypothesized based on studies in other cell types and thus insufficiently teased out to be able to draw conclusions regarding their role in ASM per se. Conversely, these topics represent emerging areas that could be relevant to understanding and targeting airway disease. Topics include 1) the role of TLRs in the context of infection effects at the level of ASM (143, 153, 193, 300, 340) and the related inflammasome (221, 416); 2) the role of mechanosensitive pathways that are being increasingly recognized in other diseases or aspects of lung growth and disease, including YAP/TAZ (320, 453), Wnt/β-catenin (105, 151, 212), and Piezo channels (28, 192, 430, 542); 3) the role of circadian rhythms and clock genes in the context of normal airway structure/function and asthma (145, 504, 505, 531); and 4) the role of senescence mechanisms that have been explored in other lung cell types and diseases (23, 50, 96, 159, 204, 207, 262, 276, 378) but are likely important in the context of the aging airway and asthma in the elderly.

An obvious consideration when exploring the role of ASM is the presence of surrounding cells and the ECM. Clearly, by virtue of its response to secreted factors by surrounding epithelial cells, fibroblasts, nerves, immune cells, and even to the ECM, as well as, conversely, being a source of factors that influence its surrounding, the concepts of cell-cell and cell-matrix interactions are emerging areas of interest. This is particularly relevant in the context of inflammation or other insults, where initial responses may involve immune and epithelial cells (for example) and the ASM may “simply” be a recipient, but with chronic disease, ASM may take on more complex roles including that of a synthetic and immunomodulatory cell and a source of pro- or anti-inflammatory factors. Furthermore, by contributing to remodeling including fibrosis, ASM can modulate the extent of proliferation and migration of other cell types, alter barrier function, and promote vascularity. Thus ASM may contribute to maintenance and exacerbation of airway disease even following the removal of the insults that initiate disease and therefore be a part of the disease phenotype that is resistant to current therapies that largely target inflammation. How this role of ASM differs between ages, sexes, initiating conditions for disease, and a host of other variables is not known but could be important to understanding and treating diseases such as asthma. Accordingly, it becomes important not only to understand mechanisms that contribute to ASM structure and function in health and disease, but also to develop appropriate, integrative biological, imaging, informatics, and other approaches (including those involving multiple cell types to facilitate study of cell-cell interactions) to analyze pathways and interactions and overall place ASM in the context of airway disease.

GRANTS

This research was supported by National Heart, Lung, and Blood Institute Grants HL088029, HL056470, and HL126451 (Y. S. Prakash).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the author(s).

AUTHOR CONTRIBUTIONS

Y.S.P. conceived and designed research, prepared figures, drafted manuscript, edited and revised manuscript, and approved final version of manuscript.