Airway Smooth Muscle in Contractility and Remodeling of Asthma: Potential Drug Target Mechanisms

Abstract

Introduction:

Asthma is characterized by enhanced airway contractility and remodeling where airway smooth muscle (ASM) plays a key role, modulated by inflammation. Understanding the mechanisms by which ASM contributes to these features of asthma is essential for the development of novel asthma therapies.

Areas Covered:

Inflammation in asthma contributes to a multitude of changes within ASM including enhanced airway contractility, proliferation, and fibrosis. Altered intracellular calcium ([Ca²⁺]_i) regulation or Ca²⁺ sensitization contributes to airway hyperreactivity. Increased airway wall thickness from ASM proliferation and fibrosis contributes to structural changes seen with asthma.

Expert Opinion:

ASM plays a significant role in multiple features of asthma. Increased ASM contractility contributes to hyperresponsiveness, while altered ASM proliferation and extracellular matrix production promote airway remodeling both influenced by inflammation of asthma and conversely even influencing the local inflammatory milieu. While standard therapies such as corticosteroids or biologics target inflammation, cytokines, or their receptors to alleviate asthma symptoms, these approaches do not address the underlying contribution of ASM to hyperresponsiveness and particularly remodeling. Therefore, novel therapies for asthma need to target abnormal contractility mechanisms in ASM and/or the contribution of ASM to remodeling, particularly in asthmatics resistant to current therapies.

I. Introduction

Asthma is a major public health concern affecting an estimated 339 million people worldwide across the age spectrum [1]. In Western countries, asthma is a leading cause of missed work and school, contributing to significant medical, economic, and social costs [2]. In spite of substantial research towards understanding the pathophysiology of asthma, and the development of a range of established and novel therapeutic approaches, a significant proportion of asthmatics show poor control, highlighting the need for identifying currently untargeted mechanisms of asthma, and development of novel alleviating strategies.

We now recognize that asthma represents a complex network of interconnected genetic, cellular, tissue/organ, and humoral factors modulated by environmental exposures, lifestyle, and in the context of continued disease, adherence to therapy, all of which make it difficult to identify singular mechanisms or therapeutic targets [3–5]*. Nonetheless, established and emerging research points to appealing approaches to asthma that are explored in this review.

The hallmarks of asthma include airway inflammation with airway hyperresponsiveness (AHR) and airway remodeling leading to overall airway obstruction [6,7]. While there is no doubt that a multitude of immune cells (considering that asthma is a disease of inflammation) and also resident airway cell types contribute to the disease, there is now considerable evidence that airway smooth muscle (ASM) significantly contributes to the pathophysiology of asthma by modulating several features including contractility, proliferation, migration, production of extracellular matrix, and even expression of inflammatory mediators that modulate the local environment within the asthmatic airway [8–11].

ASM is the key cell type responsible for generation of airway tone and contraction [8]. Upon bronchoconstrictor agonist stimulation, an increase in intracellular Ca²⁺ initiates a signaling cascade involving binding of Ca²⁺ and calmodulin, myosin light chain kinase (MLCK) activation, and actin-myosin interactions leading to contraction [12]. Dynamic regulation of intracellular Ca²⁺ ([Ca²⁺]_i) in response to different stimuli is a complex process. Therefore, dysregulation of pathways regulating [Ca²⁺]_i can contribute to the increased contractility of asthma [13–15]. During the past two decades, substantial research towards understanding mechanisms that lead to a hypercontractile phenotype of asthmatic ASM or the failure of current bronchodilatory or other therapeutics has been conducted in human ASM cells and tissue models, pre-clinical animal models of allergic asthma, and even in humans [16–18]. In this review, the literature is evaluated regarding mechanisms of ASM hypercontractility in the context of inflammation which may serve as a basis for identifying potential drug targets. Figure 1 provides a summary of suggested contributing pathways.

Figure.1: Pathways involved in airway hypercontractility:

The figure displays key pathways upregulating intracellular Ca²⁺ homeostasis and Ca²⁺ sensitivity. Proinflammatory mediators such as TNFα, growth factors (TGFβ), IL-4, IL-13, and Alarmins (TSLP, IL-33, IL-25) and external factors such as allergens, pollutants, and cigarette smoke activate different components ( GPRC, ROC, CD38, CaSR, Orai1, SOCE, and Caveolae)) within the asthmatic airway smooth muscle cells (ASM) where it produces secondary messengers bind to its receptors (IP3R, RYR, and STIM) on the sarcoplasmic reticulum (SR) to facilitate Ca²⁺ release. The Ca²⁺ -calmodulin also stimulates myosin light-chain kinase (MLCK) to initiate muscle contraction. Created with BioRender.com.

II. Hallmarks of asthma

Asthma is characterized by inflammation, AHR, remodeling and variable airway obstruction.

Inflammation

Airway inflammation is a key driver in multiple aspects of asthma pathophysiology and is thought to involve either T-helper cell type 2 (Th2; type 2) or non-Th2 (non-type 2) mechanisms [19–21]. Airway inflammation is triggered by a variety of allergens such as pollen, grass and hay, house dust mites, and even food products, which trigger a TH2 response leading to the stimulation of eosinophils, neutrophils, mast cells, and basophils [9,22]. These immune cells infiltrate the airways and regulate pathological changes via the release of a series of different pro-inflammatory mediators which play a role in providing a microenvironment for the maturation and recruitment of other immune cells and sustained inflammation [22–24].

While our understanding of the immunobiology of asthma is constantly evolving, TH2^HIGH airway inflammation is seen as the most encountered inflammatory pattern in asthma with predominantly eosinophilic inflammation and increased levels of traditional TH2 cytokines IL-4, IL-5, IL-9, and IL-13 which activate a series of downstream processes [25] leading to reduced lung function (in severe cases) and increased rates of asthma exacerbations [26]. IL-4 is a key regulatory cytokine in the development of allergic asthma with pleiotropic effects. It is associated with induction of the ε isotype switch and secretion of IgE by B lymphocytes, the ability to drive differentiation of naive T helper type 0 (T_H0) lymphocytes into T_H2 lymphocytes, and preventing apoptosis of T lymphocytes [27,28]. IL-5 plays a central role in production, maturation, and release of eosinophils in bone marrow, and regulates their tissue trafficking activation and survival [29,30]. IL-9 is known to govern the growth and differentiation of mast cells [31,32]. IL-13 is considered to be a central mediator of allergic asthma [33]. IL-13 alone is sufficient to induce responses in murine models of allergen challenge that resemble human asthma. IL-13 and IL-4 receptor (IL4R) polymorphisms are associated with asthma susceptibility. IL-13 triggers macrophage and eosinophil activation to accelerate airway inflammation, and IgE production by B cells. IL-13 contributes to AHR, enhanced and altered mucus secretion and growth factor production in airway epithelial cells, eotaxin production to stimulate eosinophil recruitment to the airways, and activation/ proliferation/ migration of airway fibroblasts to promote airway remodeling [33]. Thus, there is substantial interest in the targeting of IL-13 pathways towards alleviating asthma (e.g., dupilumab).

TH2 inflammation can also be initiated in response to alarmin production by the epithelium following exposure to different stimuli such as inhaled pollutants and microbes known to exacerbate asthma [34]. The best-investigated alarmins in asthma pathogenesis using murine models and human tissue include thymic stromal lymphopoietin (TLSP), IL-33, and IL-25 which have been all shown to activate the production of TH2 cytokines leading to airway inflammation and AHR [35,36]. There is now substantial interest in the targeting of TSLP in asthma with the approval of tezepelumab [37].

Non-allergic eosinophilic asthma mostly develops in the absence of an allergen-dependent activation of TH2 lymphocytes and patients with non-allergic eosinophilic asthma often do not respond to treatment with inhaled corticosteroids [11]. ILC2s (type 2 innate lymphoid cells) have been implicated in non-atopic eosinophilic asthma. Recent research showed that these cells are key mediators in the production of TH2-associated cytokines including IL-5 and IL-13 upon stimulation with TSLP and IL-33 [38]. There is evidence that ILC2s play a significant role in the pathogenesis of asthma, as evidenced by the increase of ILC2s in asthmatic peripheral blood [39], and sputum [40]. It is believed that these cells are critically involved in the development of type 2-mediated airway inflammation during viral-induced asthma exacerbations [41]. In addition these cells have been shown to promote AHR in mice that was prevented by inhibition of vascular endothelial growth factor (VEGF) signaling [42].

There is a predominant role for non-eosinophilic TH1 and TH17 pathways as mediators of TH2-low inflammation that may be neutrophilic or non-neutrophilic [43,44]. A number of cytokines including IL-17A, IL-17F, IFN-γ, and IL-22, are expressed by TH1, TH17, and ILC3 cells, which in turn enhance expression of a number of other pro-inflammatory cytokines and chemokines such as CXCL10, IL-6, CXCL8, and GM-CSF, primarily produced by neutrophils, but also vascular endothelial cells, fibroblasts, eosinophils, and epithelium [45]. Patients with mild to moderate asthma have higher levels of IL-17A in bronchial submucosa and bronchoalveolar lavage fluid than healthy non-asthmatics, although this is not the case in severe asthmatics [45,46]. A transcriptomic analysis of immune cells and sputum has identified an increased number of neutrophils as well as type 17 inflammation among individuals with severe asthma who do not have the TH2^high endotype [47,48].

TH1 cells produce tumor necrosis factor-alpha (TNF-α) which is an important cytokine in the pathogenesis of asthma [49]. Studies in sensitized rats have shown that TNFα exposure causes AHR and inflammation while exposure to lipopolysaccharide (LPS) increases TNF-α concentrations in the bronchoalveolar lavage (BAL) fluid [50]. In addition, TNF-α has been shown to induce a hypercontractile phenotype in human ASM cells by modulating the expression of other cytokines or chemokines and further enhancing [Ca²⁺]_i [13,51–53]. IFNγ is another TH1 cytokine known to have substantial pro-inflammatory effects on ASM in contributing to inflammation, remodeling, and AHR [43,54]. Interestingly, exposure to both TNF-α and IFNγ causes corticosteroid insensitivity in human fetal and adult ASM [55,56].

Overall, as briefly summarized above, the inflammatory environment of asthma is complex, and likely varies with induction vs. maintenance of disease, with different inflammatory mediators playing differential roles in different airway compartments, leading to a variety of changes in airway structure and function. Thus, from a therapeutic perspective, if inflammation is the sole driver of asthma, then anti-inflammatory approaches such as corticosteroids, or the newer biologics that target asthma-relevant inflammatory mediators should be uniformly effective but are not. Accordingly, there is increasing interest in the idea of targeting resident cells of the airway such as epithelium, ASM and fibroblasts and even the nervous system given these cell types could take on immunomodulatory roles as well as have effects on AHR and remodeling independent of the immune system. In this review we focus on ASM as one such key cell type.

Airway Remodeling

Airway remodeling in asthma represents the structural changes in airway walls believed to be a consequence of the repeated cycle of injury and repair [8,57]. It is generally assumed that these changes are largely irreversible [58], hence are indicative of disease progression.

Among the factors associated with airway remodeling are increased smooth muscle mass, angiogenesis, subepithelial fibrosis in the basement membrane layer below the epithelium, and epithelial metaplasia, which results in epithelial fragility and increased mucus secretion by goblet cells [8,9,59]. Airway remodeling is strongly correlated with inflammation, but certain structural change can occur independent of inflammation, such as mechanical stresses resulting from bronchoconstriction [60,61]. Abnormal deposition of the ECM in the lamina propria, submucosa, and reticular basement membrane region contributes to airway obstruction and airway wall thickening [62]. A number of ECM proteins are increased in asthmatic airways, including adhesion molecules tenascin, fibronectin, and structural collagens (I, III, and V) in addition to lumican and biglycan [63–65]. The expression of ECM molecules by ASM, fibroblasts, and epithelial cells appears to be stimulated by external factors such as viruses, cigarette smoke, and biomass fuels [66]. Additionally, asthmatic airway epithelial cells stimulate lung fibroblasts to express fibronectin, collagen, and the pro-fibrotic mediator, TGF-β [61,67]. In this regard, ASM cells exhibit an increased expression of ECM components such as fibronectin and collagen [18,68,69] making them a key cell type to target in the context of limiting remodeling. ECM proteins are degraded by proteolytic enzymes known as matrix metalloproteinases (MMPs) which in turn are inhibited by tissue inhibitors of metalloproteinases (TIMPs). Although abnormalities in both MMPs and TIMPs are reported in asthma, little progress has been made in understanding the exact role that ECM remodeling via these components plays in this disease [70–72].

Airway remodeling involves an increase in ASM mass resulting from hyperplasia and possibly hypertrophy [73,74]. Changes in the ASM phenotype from contractile to more proliferative along with generation of ECM and even inflammatory mediators (i.e. synthesis) is a key switch in asthmatics [8]. Whether ASM undergoes hyperplasia or hypertrophy (a debated issue), the resultant increase in ASM mass allows for a greater airway contractility as well as thickening in response to contractile agonists, and thus greater narrowing of the airways [75]. Thus increased ASM mass contributes both directly and indirectly to AHR in asthma.

ASM cells contribute to the airway remodeling process via the release of several growth factors such as TGFβ which has been shown to act in an autocrine and paracrine manner to initiate the differentiation and proliferation of ASM cells, regulate epithelial-to-mesenchymal transition (EMT) process and controls mucus hypersecretion [76,77]. TGFβ also stimulates a pro-contractile phenotype in ASM by increasing levels of smooth muscle alpha-actin, a feature that could contribute to fixed airflow obstruction [78].

While the focus of this review is on ASM, it is important to highlight the role of other cell types. Studies have demonstrated an apoptotic function of epithelial cells which is dependent on paracrine factors like TGF-β leading to loss of tight junction proteins, aberrant tissue repair, and remodeling of the airway [79]. Mucus-related abnormalities including mucus hypersecretion, increased goblet cell number, and enlarged mucus glands are features of airway remodeling [59]. MUC5AC and MUC5B are known to be increased in asthmatic patients and contributing to AHR, airway resistance, mucus plugging, and impaired lung function [80]. Subepithelial fibrosis has been reported in both children and adults with asthma while basement membrane thickness has been linked to TH2 inflammatory cells and mediators [81], and is strongly associated with increased airway thickness on CT, and obstructive lung function [82–84]. Bronchial vascular remodeling is another feature characterized by the formation of new vessels, and an increase in blood flow [85]. Neoangiogenesis is mediated by vascular endothelial growth factor (VEGF) where elevated levels of VEGF are associated with severe symptoms and restricted airflow in asthma patients [86]. Finally, altered airway innervation is an entirely critical aspect of airway irritability and even remodeling, involving ASM, fibroblasts, epithelial cells, and immune cells [87]. Neurally-derived acetylcholine (ACh) may promote airway inflammation and remodeling, including excessive deposition of ECM [88] and ASM mass [89]. Overall, these aspects highlight the complexity of airway remodeling and the many roles of different resident cell types of the airway independent of immune cell function. Thus, any therapeutic alleviation of remodeling will need to target these cell types and underlying mechanisms.

Airway Hyperreactivity

Airway hyperreactivity also known as airway hyperresponsiveness (AHR) is defined as an exaggerated bronchoconstrictor response to a stimulus that would have little or no effect in healthy or non-asthmatic subjects [4,90]. AHR can be triggered by direct stimuli (allergens, environmental factors) or indirect stimuli (e.g., exercise, heat, cold air) which stimulate cells such as mast cells, to release contractile agonists that induce ASM contraction [91]. AHR can be tested using direct pharmacological stimuli like histamine (H1 receptors) or methacholine (muscarinic (M3) receptors) [92]. The mechanisms underlying AHR are multiple, ranging from a contractile defect of ASM itself (increased muscle mass) in asthmatic patients and increased expression of certain contractile proteins like smooth muscle alpha-actin [78,93] to dysregulation of [Ca²⁺]_i. All these changes contribute to AHR leading to impaired airway function [8,59,94]. Inflammation in asthma increases [Ca²⁺]_i and contractile responses to agonists such as ACh [8,53] and contributes to AHR. These and other effects on contractile responses will be discussed in the next section.

III. Contractility in asthma

As mentioned previously, a primary role of ASM is to generate and maintain airway tone and regulate contraction [8,95,96]. A variety of studies using ASM from humans and different animal species have demonstrated ASM contraction and relaxation using a variety of pharmacological agonists targeting different signaling pathways [13,17,52,97] Studies using precision-cut lung slices have been informative as it allows assessment of contraction of the intrapulmonary airway and [Ca²⁺]_i signaling simultaneously [98].

Contractility involves two components, [Ca²⁺]_i regulation as well as Ca²⁺ sensitization (i.e., the amount of force produced at a constant submaximal [Ca²⁺]_i [8,99] with [Ca²⁺]_i regulation being the main driver. We and others have demonstrated that agonist-induced [Ca²⁺]_i responses are increased in ASM cells from asthmatic patients [100–102]. Increases in [Ca²⁺]_i can be initiated through two main mechanisms, increased Ca²⁺ influx via the plasma membrane or increased sarcoplasmic reticulum (SR) Ca²⁺ release [8,53,103]. Both mechanisms are capable of increasing [Ca²⁺]_i when an agonist is administered, e.g. by activating G protein-coupled receptors (GPCRs). A bronchoconstrictor agonist such as acetylcholine (ACh), histamine, or endothelin activates the PLC-IP3 pathway, causing Ca²⁺ influx and Ca²⁺ release from the SR [104]. Reduction in [Ca²⁺]_i occurs through reuptake of Ca²⁺ back into the SR via the Ca²⁺ ATPase pump SERCA, or via efflux of Ca²⁺ across the plasma membrane. The roles of some of these pathways in the asthmatic ASM are described further below

Beyond [Ca²⁺]_i, proteins regulating contractility such as MLCK are increased in sensitized animal and human airways [105], thereby contributing to AHR. Asthmatic patients show hypersensitivity to allergens, partly due to calcium sensitization in ASM cells causing increased bronchoconstriction [15,53]. There is evidence that the RhoA/Rho Kinase (ROCK) pathway is primarily promoting the activation of ASM cells and Ca²⁺ sensitization by increasing phosphorylation of MLC20 [106]. Rho kinase phosphorylates the MYPT1 myosin-binding subunit, which inhibits MLCP’s enzymatic activity, leading to prolonged phosphorylation of the myosin light chain and contraction [107]. Many mechanisms can generate ASM relaxation including the binding of the myosin phosphatase PP1 subunit to MYPT1 (myosin phosphate targeting subunit1) within MLCP [108]. in addition, cAMP formation activates PKA or cGMP which in turn activates PKG [109].

In the past, it has been discussed whether the ASM of asthmatics is fundamentally hypercontractile or the result of the inflammatory environment. The results have been largely conflicting. Some studies demonstrated increased shortening velocity of asthmatic ASM [110], while other studies reported an increased force generation in asthmatic ASM, and others found no difference compared to healthy controls [111–113]. None of these studies examined the biological effects of inflammation and cytokines on ASM contraction at a cellular level [9]. Ma et al. presented the first evidence of ASM hypercontractility in asthma at the cell level showing an increased expression of smMLCK-108 mRNA using asthmatic smooth muscle cells [114]. Data from several studies using gel contraction assays demonstrated increased agonist-induced contraction in asthmatic ASM cells compared to non-asthmatic controls [115]. Expression of the contractile protein α-smooth muscle actin in asthmatic ASM correlates with the severity of AHR [116], providing further evidence for an intrinsic change of ASM function in the asthmatic airway.

IV. Pathways involved in inflammation and hypercontractility

Investigating the possible pathways involved in ASM hypercontractility is challenging and complex. Multiple components can contribute to hypercontractility or bronchoconstriction in asthmatic patients. In addition, the observed changes can occur at different levels, e.g. protein phosphorylation level, or gene expression [59]. Although not all the mechanisms driving hypercontractility are currently understood, several pathways have been suggested.

Abnormalities in Calcium Homeostasis

Studies indicate that the inflammatory milieu in asthma pathogenesis alters the contractile phenotype of ASM, where asthmatic ASM demonstrates sustained [Ca²⁺]_i influx in response to contractile agonists, increased SR Ca²⁺ release, and a decrease in SR Ca^2+-ATPase (SERCA) activity, overall increasing [Ca²⁺]_i levels [117,118]. Intracellular pathways involve inositol triphosphate receptors (IP3Rs), ryanodine receptors (RyRs) and SERCA [13,100,119–122]. Thus, these mechanisms represent potential targets to reduce AHR.

While classical intracellular Ca²⁺ release via the endoplasmic reticulum in a number of cell types involves IP3Rs, the CD38/ cyclic adenosine diphosphate ribose (cADPR)/ ryanodine receptor (RyR) pathway has been shown to affect [Ca²⁺]_i homeostasis in airways [123]. Animal studies have demonstrated a reduction in AHR in CD38-deficient mice (CD38 KO mice) [124]. A variety of cytokines such as TNF-α, IL-1β, IL-13, and IFN-γ was found to increase the expression of CD38 as well as cADPR activity and [Ca²⁺]_i after stimulation of various contractile agonists in human ASM [97,120,125,126] Moreover, TNF-α has shown to significantly increase CD38 in ASM cells derived from asthmatic donors compared to controls [123]. Overexpression of CD38 was also observed in ASM cells obtained from patients who died from asthma episodes or patients with stable asthma in both transcriptional and protein levels compared to non-asthmatics [127]. Thus, there is substantial interest in targeting the CD38 pathway in reducing AHR.

Store-operated calcium entry (SOCE) is responsible for refilling of the SR via Ca²⁺ influx from the extracellular environment following SR depletion. SOCE plays a role in asthma remodeling and AHR [128,129]. In the context of SOCE, the stromal interaction molecule 1 (STIM1) located on the membranes of the ER and SR serves as a Ca²⁺ sensor [130,131]. It has been shown that the expression of STIM1 in ASM cells isolated from ovalbumin-challenged asthmatic mice is essential for the proliferation and migration of ASMs [132], while STIM1 drives AHR and remodeling in asthmatic mice through enhanced frequency and amplitude of ASM cytosolic Ca²⁺ oscillations [133]. We showed that plasma membrane invaginations called caveolae are present in human ASM and that the regulatory protein caveolin-1 promotes [Ca²⁺]_i responses to agonists [100]. Furthermore, TNF-α increases the expression of caveolin-1 and Orai1 (the key plasma membrane channel for SOCE that interacts with STIM1), resulting in enhanced interaction between the plasma membrane (Orai1) and the SR, thereby facilitating SOCE [134]. Thus, modulation of SOCE represents a novel target in asthma.

Previous research including our own has demonstrated that factors such as cigarette smoke which exacerbate asthma can impact on ASM. We demonstrated that cigarette smoke extract alters [Ca²⁺]_i regulation in human ASM cells via increases in baseline [Ca²⁺]_i, agonist-induced [Ca²⁺]_i responses, and increased SOCE [125]. Here, increased expression of CD38, transient receptor potential canonical 3 (TRPC3), Orai1 and STIM1 has been noted [8,100,134].

Another contributor to airway inflammation and AHR is the extracellular calcium-sensing receptor (CaSR) [135–137]. CaSR is the regulator of extracellular Ca²⁺ concentration and highly expressed in all organs involved in the control of mineral ion metabolism [138]. Signaling via the CaSR has also been reported to be upregulated in fetal lung ASM suggesting a role for CaSR in AHR in premature infants ventilated with supplemental oxygen [137]. In addition, CaSR plays a role in airway inflammation as it is highly expressed in most of the inflammatory cells involved in asthma pathogenesis e.g. monocyte/macrophages, neutrophils, T cells, and eosinophils, which is believed to lengthen their lifecycle by inhibition of apoptosis [135,139]. A recent study of genetic variants of CaSR shows a clinical association with several diseases including asthma [140]. We have previously shown that CaSR is highly expressed in adult human ASM of asthmatics and contributes to both increased [Ca²⁺]_i regulation (working via intracellular release) as well as cell proliferation [135]. Furthermore, mice constitutively lacking CaSR show reduced AHR and remodeling in response to allergens [135]. Thus, CaSR may represent a novel target in AHR of asthma.

Abnormalities in Calcium Sensitivity and Sensitization

As discussed previously, smooth muscle contraction is dependent on the interaction of calcium/calmodulin and MLCK. Increased [Ca²⁺]_i activates phosphorylation of MLC20 and subsequent contraction of ASM [141]. An elevated MLCK mRNA and increased shortening velocity are observed in ASM cells from asthmatic patients [114]. In addition, TGF-β1 exposure augments bronchoconstriction in human lung tissue slices, with a Smad3-ROCK-dependent pathway being responsible for this effect by increasing MYPT1 phosphorylation [142].

Animal models of coronary artery spasm and hypertension exhibit abnormal RhoA/Rho-kinase pathway activity [143]. The same finding has been reported in an animal models of allergic asthma [144] and in asthmatic human lung [145]. RhoA/Rho-kinase signaling is key to Ca²⁺ sensitization (Ca²⁺ independent) where it acts by removing phosphate from the phosphorylated MLC20 to cause ASM relaxation [146,147]. Protein expression of RhoA is increased in bronchial smooth muscle derived from rat models of allergic asthma [144]. Furthermore, asthma-associated cytokines (IL-13, IL-17, and TNF-α) increase the expression of RhoA and CPI-17 (C-potentiated phosphatase Inhibitor) which binds to the catalytic subunit of MLCP, inhibiting its phosphatase activity in animal and human ASM cells [148], demonstrating the involvement of the RhoA pathway in AHR and airway remodeling.

Abnormalities in Airway Innervation

Existing research recognizes the critical role of nerve activity driving AHR seen in asthma [149,150]. There is increasing interest in the role of nicotinic acetylcholine receptors (nAChRs) in driving AHR and airway inflammation in asthma. Among the nAChRs subtypes expressed in human ASM, α7-nAChR is the one most abundantly expressed [151]. Ca²⁺ entry via α7-nAChR has been shown to modulate several Ca²⁺ dependent cellular processes [152]. Moreover, nicotine-induced proliferation of ASM cells is mediated by α7-nAChR [153]. Recently, we showed that human ASM expresses α7-nAChR which is increased in inflammation suggesting a role in AHR and other functional changes that occur with nicotine or cigarette smoke exposure for example that may be relevant to asthma [154].

Nerve-derived factors such as neurotrophins (NT) and neurotrophin receptors (NTR) are expressed in postganglionic non-adrenergic non-cholinergic (NANC) nerves [155]. Increased expression of nerve growth factor (NGF) has been found in the serum of asthmatic patients [156]. Expression of brain-derived neurotrophic growth factor (BDNF) and tropomyosin-related kinase (TrkB) have also been associated with asthmatic airways [155,156]. In vitro studies in human ASM show enhanced [Ca²⁺]_i responses to ACh, histamine, and bradykinin after exposure to BDNF and other NTs [52] which is further enhanced in the presence of TNFα [49]. Exposure to BDNF also increases SOCE in human ASM [52]. Thus, growth factors such as the NTs may represent entirely novel targets for AHR.

V. Potential drug targets

Inhaled corticosteroids (ICSs) and β2-adrenergic receptor agonists remain mainstays of asthma therapy [157] and are clinically effective in most patients [158]. However, some asthmatic patients remain at high risk of severe exacerbation, hospitalization, and mortality [159]. Anti-inflammatory agents or anticholinergics indirectly target smooth muscle cell dysfunction [160]. Bronchial thermoplasty targets ASM directly by reducing smooth muscle mass although the mechanisms of action and long-term side effects are still unclear [161]. Monoclonal antibodies such as omalizumab, mepolizumab, benralizumab, dupilumab, and tezepelumab have been approved by the FDA and given their targeting of key inflammatory pathways such as IL4, IL-13 and TSLP, they should be effective in modulating ASM contractility in the context of AHR [162]. Whether they will be as effective in targeting remodeling remains less clear given (as summarized briefly earlier) the contribution of different inflammatory pathways in different cell types change during the course and severity of disease. Thus, the field remains open for targeting of non-immune or non-inflammation pathways in asthma. Here, we believe the ASM (among other cell types) is an appealing avenue.

AHR has been suggested to be a main treatable characteristic towards developing precision medicine in patients suffering from TH2 high asthma, therefore there is an unmet need for development of biological therapies for long-term disease management. Table1 summarizes potential targets thought to have direct effect on ASM hypercontractility and AHR. Of course, in many if not all of these options, targeting the airways and ASM in particular is critical without causing systemic effects, particularly given that many if not all of the mechanisms listed are relevant to other types of smooth muscles if not other cell types across different organs. Here, targeting Ca²⁺ regulatory pathways particularly CD38 or SOCE is appealing. On the one hand there are multiple diseases in which CD38 is thought to play a role via its modulation of cell signaling and metabolism, and there is increasing interest in developing small molecule modulators of CD38 activity which when provided via the intratracheal route, could be used to directly target the bronchial airways. In a similar vein, cADPR the product of CD38 activity also holds promise given the increasing number of cADPR inhibitors that again could be provided via an intrapulmonary route to limit systemic effects. Targeting the CaSR is also within the realm of reality given ongoing phase I clinical trials in the UK and given the safety profile of CaSR modulators in the context of osteoporosis and hyperparathyroidism. In terms of Ca²⁺ mechanisms, targeting of SOCE is possible given that Orai1 is overexpressed and is a plasma membrane protein. In fact, given that Orai1 can be overexpressed in asthmatic epithelium and ASM, pharmacologic inhibition of this mechanism may be effective on multiple fronts. Targeting of SERCA is likely to be a bigger challenge and perhaps less appealing, given the critical role of this ATPase in intracellular Ca²⁺ regulation and other ER functions. Beyond Ca²⁺, targeting of Ca²⁺ sensitivity and sensitization is also appealing given the increasing interest in targeting RhoA/ROCK and small Rho GTPases in the context of cancers and vascular disease. Again, intrapulmonary administration might help limit systemic effects.

Table1.

Potential future targets with direct effects on ASM aiming to reduce hypercontractility.

Potential target	Mechanism of action	Modulators	Type of Evidence	References
DGK	MLCP dephosphorylation ↑PKC	Diacylglycerol Kinase inhibitor	Animal studies Human bronchial cells	[163–165]
PLC	↓PLC	Inhibitors of phospholipase C	Animal studies Human bronchial cells	[164]
Rho Kinase	Calcium sensitivity pathway MLCP dephosphorylation ↓PGD2	Clostridium botulinum C 3 exoenzyme (RhoA inhibitor) Y-27632 (ROCK Inhibitor) HA‐1077 (RhoA inhibitor)	Animal studies Human bronchial cells	[143,145,147]
CaSR	Block CaSR	Calcilytics	Animal studies Human bronchial cells	[136,166,167]
NOX4	↓ROS	NOX4 inhibitors	Animal studies Human bronchial cells	[115]
CD38/cADPR	↓[Ca2+]i	Small molecule CD38 inhibitors	Animal studies Human bronchial cells	[97,168]
STIM1	↓[Ca2+]i oscillations	Thapsigargin	Animal studies Human bronchial cells	[129]
SERCA2	SERCA dysfunction	Gene therapy	Animal studies Human bronchial cells	[118]
nAChRs (α7-nAChR)	↓[Ca2+]i	α7-nAChR inhibitors	Human bronchial cells	[154]
NGF	↓(RhoA)–ROCK pathways	NGF inhibitors	Animal studies Human bronchial cells	[150,169,170]
Gq-coupled G protein-coupled receptor	↓Gq-mediated signaling pathways	Gq protein inhibitor FR900359 (FR)	Animal studies	[171]
TAS2R	Activation of BK channel ↓VDCC ↓ IP3R	TAS2R agonists (Chloroquine, Quinine)	Animal studies Human bronchial cells	[172–174]
GABA receptors	↑cAMP ↓IP3R ↓SERCA	GABAR Agonist (MIDD0301)	Animal studies ASM cells	[175–177]
MAP kinase inhibitor	↓MAPK markers (P38, JNK, ERK…)	p38 MAPK inhibitors (SB239063) MAPK kinase inhibitor (U0126) JNK inhibitor (SP600125) ERK1/2 inhibitor (SF-3–030)	Animal studies ASM cells	[178]

MLC: Myosin Light Chain. Rho Kinase: Rho-associated protein kinase. CaSR: calcium-sensing receptor. NOX4: nicotinamide adenine dinucleotide phosphate oxidase type 4. CD38: cluster of differentiation 38. cADPR: Cyclic ADP Ribose. STIM1: Stromal interaction molecule 1 SERCA: sarco/endoplasmic reticulum Ca2+-ATPase. nAChRs: Nicotinic acetylcholine receptors. α7-nAChR: alpha-7 nicotinic receptor NGF: Nerve growth factor, TAS2R: Bitter-taste receptors, GABA receptors: Gamma-Aminobutyric Acid, MAPK: Mitogen-activated protein kinases

Novel approaches beyond the contractility of AHR involving ASM per se involve growth factors which could be targeted akin to the biologics that target inflammatory pathways. Given these are locally secreted within the airway, intrapulmonary administration of chelators such as TrkA-Fc TrkB-Fc (targeting NGF and BDNF) represent a novel way to limit the sustained effects of NTs beyond initial inflammatory insults. Targeting them limits only on AHR but also airway remodeling.

VI. Conclusion

Airway hyperreactivity and remodeling are now recognized as key features of asthma. The mechanisms and processes leading to AHR, and remodeling are complex and not fully understood. Addition of new environmental factors like vaping have added to the challenges of understanding and treating asthma. Over the past few years, academia and industry have intensified efforts to develop new treatments targeting airway inflammation in particular with indirect effects on AHR and less so remodeling. These approaches allow for extension of asthma therapies beyond β2-adrenergic receptor agonists, anticholinergics, and corticosteroids, but with data on steroid-resistant asthma and its consequences, the time is now to focus on new cell types and mechanisms to limit AHR and particularly remodeling. Here, we propose the targeting of ASM with potential for pleiotropic effects in alleviating asthma.

Article Highlights.

• Despite therapeutic advances, asthma significantly impacts patients’ lives and overall healthcare

• Increased insight into mechanisms contributing to asthma has broadened therapeutic options and allowed for more goal-oriented therapies

• Therapeutic advances of drug delivery have broadened the potential targets of drug delivery and allowed for improved combined therapeutics

• These advances have improved treatment options for asthmatics but also demonstrated future needs