Abstract
BackgroundMesenchyme-derived airway cell populations including airway smooth muscle (ASM) cells, fibroblasts and myofibroblasts play key roles in the pathogenesis of airway inflammation and remodeling. Phenotypic and functional characterisation of these cell populations are confounded by their heterogeneity in vitro. It is unclear which mechanisms underlie the creation of these different sub-populations.The study objectives were to investigate whether ASM cells are capable of clonal expansion and if so (i) what proportion possess this capability and (ii) do clonal populations exhibit variation in terms of morphology, phenotype, proliferation rates and pro-relaxant or pro-contractile signaling pathways.MethodsEarly passage human ASM cells were subjected to single-cell cloning and their doubling time was recorded. Immunocytochemistry was performed to assess localization and levels of markers previously reported to be specifically associated with smooth muscle or fibroblasts. Finally functional assays were used to reveal differences between clonal populations specifically assessing mitogen-induced proliferation and pro-relaxant and pro-contractile signaling pathways.ResultsOur studies provide evidence that a high proportion (58%) of single cells present within early passage human ASM cell cultures have the potential to create expanded cell populations. Despite being clonally-originated, morphological heterogeneity was still evident within these clonal populations as assessed by the range in expression of markers associated with smooth muscle cells. Functional diversity was observed between clonal populations with 10 μM isoproterenol-induced cyclic AMP responses ranging from 1.4 - 5.4 fold cf basal and bradykinin-induced inositol phosphate from 1.8 - 5.2 fold cf basal.ConclusionIn summary we show for the first time that primary human ASM cells are capable of clonal expansion and that the resulting clonal populations themselves exhibit phenotypic plasticity.
Highlights
Mesenchyme-derived airway cell populations including airway smooth muscle (ASM) cells, fibroblasts and myofibroblasts play key roles in the pathogenesis of airway inflammation and remodeling
In vitro phenotypic plasticity has been demonstrated as being tightly regulated: growth factors, fibronectin, collagen type I, integrins and adhesion molecules are observed to induce a synthetic phenotype whereas serum deprivation, Transforming Growth Factor β (TGF-β) and insulin are observed to induce a contractile phenotype
Given the phenotypic heterogeneity which ASM cells can exhibit in vitro [11], we aimed to investigate the clonal origin of populations of cells in ASM in order to determine whether single cells were capable of clonal expansion and to define how different resulting clones might be from each other and their parental cell in terms of morphology and function
Summary
Mesenchyme-derived airway cell populations including airway smooth muscle (ASM) cells, fibroblasts and myofibroblasts play key roles in the pathogenesis of airway inflammation and remodeling. In addition to forming the muscle mass which causes bronchospasm in asthma exacerbations, human ASM cells mediate inflammation via cytokine release [1]. Whilst in vivo little is known of the clinical relevance of these mechanisms, in vitro the ASM cell signaling pathways key to these events have been extensively researched and many pro-proliferative, pro-apoptotic and pro-migratory mediators identified [7]. In addition to these molecules, recent evidence demonstrates the ability of bronchoconstriction itself to induce airway remodeling both in guinea-pigs [8] and humans [9]. In vitro phenotypic plasticity has been demonstrated as being tightly regulated: growth factors, fibronectin, collagen type I, integrins and adhesion molecules are observed to induce a synthetic phenotype whereas serum deprivation, Transforming Growth Factor β (TGF-β) and insulin are observed to induce a contractile phenotype (see [10])
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