Clonally expanded human airway smooth muscle cells exhibit morphological and functional heterogeneity

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.

Reply to Letter to the Editor: “Bnip3 as a potential target to treat airway smooth muscle remodeling in asthma?”

“…The doubling time for human ASM cells isolated from healthy non-asthmatic donor lungs varies from 36 to 52 h as reported in previous studies (7,12,15). Rate of proliferation of ASM cells within a culture shows high degree of variability (17). Further, it has been shown that primary human ASM cells exhibit heterogeneity that dictates many functional outcomes (to both endogenous and exogenous stimuli) such as cell proliferation, growth, calcium-, chemokine-, cytokine-, extracellular matrix (ECM)release (4,6,15).…”

p116Rip promotes myosin phosphatase activity in airway smooth muscle cells

“…Although ASMCs exhibits heterogeneous properties (Ma, Li, & Stephens, ; Singh, Billington, Sayers, & Hall, ), it is likely that changes in the phenotype of ASMC occur in the disease progression of asthma. This view is supported by in vivo observation that the degree of airway responsiveness is variable depending on the severity of asthma symptoms (Busse, ).…”