Functional characterization of serum‐ and growth factor‐induced phenotypic changes in intact bovine tracheal smooth muscle

Abstract

1 The present study aims to investigate whether phenotypic changes, reported to occur in cultured isolated airway smooth muscle (ASM) cells, are of relevance to intact ASM. Moreover, we aimed to gain insight into the signalling pathways involved. 2 Culturing of bovine tracheal smooth muscle (BTSM) strips for up to 8 days in the presence of 10% foetal bovine serum caused a time-dependent (t 1/2 =2.8 days) decrease in maximal contraction (E max ) to methacholine compared to serum-deprived controls (E max =74+4% at day 8). A reduced E max was also found using insulin-like growth factor-1 (30 ng ml 71 ) and platelet-derived growth factor (30 ng ml 71 ), but not using epidermal growth factor (10 ng ml 71 ) (E max =83+3, 67+8, 100+4%, respectively). Similar serum and growth factor-induced changes in E max were found for KCl-induced contraction (65+9, 80+7, 64+11% and 107+2%, respectively). 3 Strong correlations were found between the growth factor-induced reductions in E max and their proliferative responses, assessed by [ 3 H]-thymidine-incorporation, in BTSM cells. (r=0.97, P=0.002 for methacholine and r=0.93, P=0.007 for KCl). 4 The PDGF-induced reduction in E max was inhibited completely by combined treatment with either PD 98059 (30 mM) or LY 294002 (10 mM). 5 These results indicate that serum and growth factors may cause a functional shift towards a less contractile phenotype in intact BTSM, which is associated with their proliferative response and dependent on signalling pathways involving the mitogen-activated protein kinase pathway and the phosphatidylinositol-3-kinase pathway.

Extracellular matrix proteins differentially regulate airway smooth muscle phenotype and function

“…Therefore, we assessed the expression of sm-myosin, calponin, and sm-␣-actin in homogenates prepared from BTSM strips treated with vehicle, collagen I, fibronectin, or laminin in the absence or presence of PDGF for 4 days. In accordance with our previous (8,9) and current observations that PDGF induces a shift towards a more proliferative (hypocontractile) phenotype, the growth factor reduced the expression of all contractile markers studied. Incubation with fibronectin or collagen I resulted in a reduction of contractile protein expression as well, which was both quantitatively and qualitatively similar to that induced by PDGF.…”

“…In the present study, we found a strong correlation between the degree of change of E max , both with methacholine and KCl, induced by the applied ECM proteins in BTSM strips and the proliferative response by BTSM cells cultured on these proteins. Although determined using different experimental parameters measured in different (ASM tissue and cellular) conditions, this correlation was very striking and highly reminiscent of our previous findings (8). Together with the relationship between contractile protein expression and contractility found for the applied ECM proteins, these results indicate that ECM proteins are importantly involved in the regulation of ASM phenotype and function.…”

“…It has been established that there is a strong inverse relationship between the effects of peptide growth factors on maximal methacholine-and KCl-induced contraction of BTSM strips and the proliferative response of BTSM cells to these growth factors (8). In the present study, we found a strong correlation between the degree of change of E max , both with methacholine and KCl, induced by the applied ECM proteins in BTSM strips and the proliferative response by BTSM cells cultured on these proteins.…”

“…It has been previously established that growth factor-induced changes in maximal contractility of BTSM strips are inversely correlated with changes in the proliferative response of isolated BTSM cells (8). As illustrated by Fig.…”

“…Importantly, in contrast to skeletal myocytes and cardiomyocytes (17,19), ASM cells maintain the ability to reenter the cell cycle. Thus exposure to mitogenic stimuli (e.g., PDGF) results in the induction of a more proliferative/synthetic phenotype (13), which is accompanied by a loss of contractile responsiveness (8), presumably as a consequence of decreased contractile protein expression (13). Long-term serum deprivation results in the reinduction of a contractile phenotype, underlining the reversible nature of ASM phenotype (18).…”