The ASM thickening has been studied in many animals and human models, wherein ASMC cultures have provided some ideas about pathways underlying the origin of hyperplasia and hypertrophy. Once ASMC populations were characterized in vivo and in vitro, heterogeneous subgroups with distinctive phenotypes were identified. A wide range of functions depend on culture conditions⁽⁶⁰⁾. Manipulating such environments allowed comprehension of the rules for phenotypic transition^{(61, 81, 82)}. Hence, ASMC could be sorted into three categories: 1) contractile (c-ASMC), 2) synthetic/proliferative (s/p-ASMC), and 3) hypercontractile (h-ASMC) (see Table 1). Also, a few switching routes have been described, where modulation means a shift from contractile to synthetic/proliferative, and maturation is the inverse transition. Turning into hypercontractile is also possible, and some authors have speculated about its irreversibility; however, in vitro ASMCs can tolerate cyclic phenotypic adjustments. An important aspect is that modulation and maturation exemplify an adaptation model to tissue microenvironment fluctuations, events that could take place in vivo and drive critical phases of airway remodeling. Accordingly, transition from native c-ASMC to s/p-ASMC would be the initial step, then, replication of s/p-ASMC would warrant smooth muscle hyperplasia, and finally aberrant differentiation from either s/p-ASMC or c-ASMC to h-ASMC would cause muscle hypertrophy. How these phenotypical modifications fit in the natural history of airway diseases is a matter of debate.

Phenotypic Markers

Smooth muscle has typical features in primary cultures (see Table 1, Fig. 3). A long cellular body, central nucleus, few granulations, and cytoplasmic inclusions (3A), a confluent monolayer with “hill and valley” aspect (3C), and shrunk reaction to contractile agonists define smooth muscle cells⁽⁸³⁾. However, each ASMC subpopulation has specific characteristics. For example, the synthetic/proliferative phenotype has satellite flattened shape with multiple extensions (3B), a high number of organelles for protein and lipid synthesis, abundant mitochondria, a higher proliferative response, decreased contractile proteins, shutdown of responses to contractile agonists, and secretion of growth factors, collagen, cytokines, bradykinin, and eotaxin^(81,84). Furthermore, modulated ASMCs show increased protein expression of fetal and non-muscle isoforms. Synthetic and proliferative functions do not correspond to different traits. Indeed, the cell distribution with synthetic activities could vary between 20 to 60%, and almost half of replicating ASMCs produce cytokines. Also, secretion can be done by non-replicating cells⁽⁸¹⁾. On the other hand, the contractile phenotype is associated with a decreased number of synthetic organelles, a stronger response to contractile agonists, increased expression of contractile and structural proteins, and an increased M₃/M₂ muscarinic receptor expression rate^(84-86). Other markers including Ca²⁺ profiles⁽⁸⁷⁾, miRNA expression⁽⁸⁸⁾, and transcription factors expression⁽⁸⁹⁾ have been useful for phenotype distinction.

Figure 3. Primary cultured of Rat ASMCs. Cells were obtained by enzymatic digestion of rat trachea and cultured in supplemented medium as previously described(122) . (A) Contractile phenotype, (ci) cytoplasmic inclusions, (n) cell nucleus. (B) When the cell population underwent to growth some cells adopted a myofibroblast-like morphology (F-s/pASMC), (m) mitosis. (C) Cell confluence of 80-90%, cell population readopted contractile morphology with a hill and valley array. Magnification 400X A, B, 100X C.

Evidence of in vivo Plasticity

Several findings support in vivo occurrence of ASMC plasticity, especially in asthma. Plasticity is a universal property of primary ASMC cultures derived from both healthy and diseased humans, and healthy and sensitized animals. Immunohistochemistry to identify contractile proteins is highly variable as well, which can reflect a broad heterogeneity of myocytes in the normal airway that is maintained in cell culture, as demonstrated by a divergent proliferative capacity⁽⁹⁰⁾. If functional features are compared, healthy or control vs asthmatic or sensitized groups, significant differences can be found. ASMCs from asthmatics or sensitized animals show more proliferative and synthetic capabilities than their physiologic counterparts, findings that are preserved despite tissue dissolution follow by cell culture^{(62, 66, 89, 91-93)}. Abnormal ASMCs could not only resemble s/p-ASMCs or arise from c-ASMCs, but also have distinctive features such as: abnormal protein synthesis⁽⁹⁴⁾, expression of odd transcription factors isoforms with a lack of response to glucocorticoids⁽⁹¹⁾, increased mitochondrial biogenesis and activity⁽⁹³⁾, abnormal calcium dynamics⁽⁹²⁾, increased CysLTR-1 leukotriene receptor expression⁽⁵⁵⁾, increased activity of promitogenic pathways⁽⁹⁵⁾, and declined of antiproliferative pathways⁽⁶⁶⁾. In consequence, an increased ASM mass may be explained by intrinsic alterations in pathological ASMCs that facilitate their proliferative and secretory activities. Asthmatic ASM produces more proinflammatory, proangiogenic, and proremodeling factors, including eotaxin, VEGF, and connective tissue growth factor (CTGF), and fewer antimitogenic factors, such as E2- type prostaglandin (PGE₂)⁽⁵⁹⁾. These would reflect deeper differences in cell populations that constitute the ASM under pathological settings, and they would likely originate from comparable modulation and maturation events on native ASMCs.

Many questions arise from the alterations observed on asthmatic or sensitized cultured ASMCs. Based on the plasticity phenomena, any transformation of in vivo ASMC phenotype which persistence depends on tissue microenvironment should not be seen in vitro because the phenotype will adjust to culture conditions. Although, cited studies do not precise whether those pathologic features are irreversible along culture passages, persistence of functional abnormalities after tissue fragmentation and culturing suggests that these cells underwent through a dysfunctional route of phenotypic modulation, which could be at least partially irreversible. In view of that, epigenetic mechanisms could be a suitable explanation to such phenotypic switch. For example, eotaxin hypersecretion by ASMCs has been related to histone H4 lysine 5 and lysine 12 acetylation at the eotaxin promoter induced by TNF-α⁽⁹⁶⁾. Other synthetic activities, such as VEGF hypersecretion, were due to a loss of a repression complex, in which a differential histone H3 lysine 9 methylation modulating Sp1 and RNA polymerase II binding to the VEGF promoter was implicated⁽⁹⁷⁾. Binding of serum response factor (SRF), a transcription factor that controls phenotypic stability, to DNA is associated with post-transcriptional histone modifications including di-methylation of lysine residues 4 and 79 on histone H3, acetylation of lysine 9 on histone 3 and acetylation of histone H4. Histone deacetylases (HDACs) have also been implicated in regulating smooth muscle replication because the HDAC inhibitor TSA can prevent cell proliferation⁽⁸⁸⁾. In in vivo, the valproic acid (HDAC inhibitor) did not affect inflammation induced by OVA challenges, but notably reduced the airway thickening including the ASM with blunting of AHR⁽⁹⁸⁾. In addition to HDAC modifications, DNA methylation can generate a specific long-term signature. For example, expression of IL-13 in the airways ensued significant changes in methylation of 177 genes, most of which were associated with a Th2 signature over resident cells⁽⁹⁹⁾. Using methylated DNAimmunoprecipitation-next generation sequencing (MeDIP-seq), it was determined that airway remodeling and AHR in house- dust- or mite-sensitized rats are related to specific methylation patterns at several TGF-β signaling-related genes⁽¹⁰⁰⁾, explaining the longevity of abnormal pro-fibrotic responses of local cells during inflammation and phenotypic persistence after tissue extraction. Unfortunately, there is currently no direct evidence of epigenetic regulation of ASMC proliferation. Moreover, whether or not DNA methylation or histone acetylation can influence phenotypic switching have to be determined as well. The contribution of miRNAs will be discussed in following sections.

Potential Sources of ASMCs

The in vivo source of ASMCs under pathological conditions is unclear. ASM may originate from increased proliferation or prolong survival of preexisting smooth muscle with proliferative and/or contractile phenotype; however they could also arise from other cell lines that could migrate into the bundles and then differentiate into ASMCs (see Fig.4). Remarkably, other airway cells may undergo to phenotypic modulation that is characterized by α-sm-actin expression and development of organelles for synthetic functions. Accordingly, mesenchyme such as fibroblasts may generate myofibroblasts, whose classical phenotypic markers are indistinguishable from s/p-ASMCs⁽¹⁰¹⁾. This fact allowed researchers to postulate a spectrum of mesenchymal plasticity (fibroblasts ↔ myofibroblasts ↔ ASMCs)⁽¹⁰²⁾. However, it does not undermine experimental findings obtained with in vitro systems, as a high proportion (~60%) of primary airway mesenchymal cultures truly correspond to primary smooth muscle⁽⁶⁰⁾.

Figure 4. Potential sources of ASMC precursors in the origin of ASM thickening. (See the text for explanation).

Potential progenitors also include true multipotent mesenchymal progenitors and stem cells, either located within the airway or derived from peripheral blood. For example, CD34⁺-CCR7⁺-Collagen 1⁺-sm-α-actin⁺ circulating fibrocytes can migrate towards ASM bundles during inflammatory challenges, and they were unresponsive to the apoptotic effects of glucocorticoids in culture⁽¹⁰³⁾. Fibrocyte migration is directed by the ASM-derived PDGF and CCL2, and at that point its co-locating induces proinflammatory activities in ASMCs^{(104, 105)}. A rare population of CD34⁺-sm-MHC⁺ peripheral mononuclear cells (known as smooth muscle progenitors) has been identified by flow cytometry in OVA-sensitized mice. A similar population seems to generate the smooth muscle in atherosclerosis; however, the study did not precise whether stem cell homing occurred into ASM bundles⁽¹⁰⁶⁾.

The airway epithelium can turn into mesenchymal cells through the epithelial-mesenchymal transition (EMT) route, which has been considered as another source of ASMCs⁽¹⁰⁷⁾, but a linage-tracing study suggests that it may just be a consequence of culture conditions and could not occur in vivo⁽¹⁰⁸⁾. In asthma, epithelial cells show fragileness due to downregulation of cell adhesion molecules, which makes EMT more likely⁽¹⁰⁹⁾. EMT is initiated by extracellular signals, such as collagen or hyaluronic acids, and by growth factors like TGF-β and EGF⁽¹¹⁰⁾. This process is modulated by bone morphogenesis proteins, and allergen exposure, which amplifies and accelerates it⁽¹¹¹⁾. Hormones have also been associated, since vitamin D attenuates TGF-β-induced expression of EMT markers⁽¹¹²⁾. Epithelial and mesenchymal cells express both type-1 and type-3 muscarinic receptors (M₁, M₃)⁽¹¹³⁾. TGF-β-induced EMT was abolished by muscarinic receptor (mAChR) antagonists and enhanced by acetylcholinesterase (AChE) inhibitors⁽¹¹⁴⁾. A positive feedback loop of autocrine and paracrine production of non-neuronal acetylcholine (ACh) and TGF-β orchestrates EMT during chronic inflammation, being a likely source of ASM.