Altered Airway Smooth Muscle Contractile Function in the Presence of Airway Epithelium and the Influence of TGF-β1

Abstract

Airway smooth muscle (ASM) contraction is a major contributor to bronchoconstriction, the narrowing of the airways observed in asthmatic airways. In vitro, ASM cells demonstrate the capacity to switch between more proliferative and more contractile phenotypes, and changes to ASM contractile function, potentially as a consequence of this phenotypic switching, may play a significant role in the exaggerated airway narrowing observed in asthma. In vivo, airway epithelial (AE) cells are topographically close to the ASM and may modulate and regulate ASM phenotype and function that could be dysregulated in asthma. One important mediator increased in asthma is TGF-β1, which influences AE cell phenotype and thus possibly affects AE cell effects on ASM. In this work, we investigated the effects of AE, with and without the influence of TGF- β1, on ASM contractile function. After examining the response of AE and ASM to TGF-β1 individually, one-way and two-way communication modes between the cell types were established using conditioned media and co-culture systems as routes of exposure, respectively. Cell stiffness and changes in cell stiffness in response to KCl-induced contraction, as well as protein expression, were used to assess changes in ASM contractile function. We found that the addition of AE-conditioned media to ASM dramatically increased ASM expression of myocardin, but interestingly this was not accompanied by increases in contractile function or in the expression of contraction-associated proteins in the ASM. In contrast, ASM cells in co-culture with AE did not show the same dramatic increase in myocardin expression observed with AE-conditioned media exposure. In both conditioned media and co- culture experiments however, we found that ASM stiffness was consistently decreased as a response to AE exposure; ASM contractility also decreased, but only via co-culture. These decreases in stiffness were also consistently accompanied by a decrease in the expression of the smMHC motor protein for both conditioned media and co-culture experiments. When TGF-β1 was added to ASM cells, calponin expression in ASM increased, independent of whether AE cells were present. Interestingly, only the addition of TGF-β1 into an AE-ASM co-culture led to the increased expression of some canonical markers for a contractile ASM phenotype, although this effect was divorced from any functional increase in ASM contractility. Overall, we show that AE cells had a relaxing effect on ASM cells and attenuated contractile function, likely due in part to the consistently suppressed expression of smMHC. These results demonstrate that AE plays an important role in regulating ASM contractile function and phenotype in culture, and that TGF-β1 may alter this AE-ASM interaction. Taken together this suggests that AE-ASM intercellular communication can play an important role in the regulation of ASM function that may be potentially altered in asthma.