ABSTRACT

Background: Airway remodeling is a repair process occurring after airway injury; its primary histopathological features are subepithelial fibrosis and smooth muscle thickening of the bronchi. These histopathological changes are considered to occur due to bronchial smooth muscle cells (bSMC) that secrete extracellular matrix (ECM) proteins, which work as chemoattractants and influence cell migration. Therefore, we examined the interaction between bSMCs and ECM proteins in vitro for understanding the remodeling process in the bronchi.
Methods: bSMCs were cultured to collect a bSMC-conditioned medium. Using the bSMC-conditioned medium thus obtained, we performed a cell migration assay, characterized β integrin expression, and identified ECM proteins and matrix metalloproteinases by western blotting and gelatin zymography, respectively.
Results: The response of bSMC migration to bSMC-conditioned medium increased with time in culture, and fibronectin (FIB) was detected as a chemoattractant for bSMCs in bSMC-conditioned medium by western blot analysis and a cell migration assay using anti-FIB antibodies. The involvement of β1 integrin in the migration of bSMCs toward FIB contained in bSMC-conditioned medium was demonstrated by inhibition of cell migration using anti-β1 integrin antibodies. Expression of β1 integrin on bSMCs was confirmed by using a β-integrin-mediated cell adhesion array. In addition, metalloproteinases detected in bSMC-conditioned medium by gelatin zymography were suggested to be matrix metalloproteinase-1 and 2 by western blotting and amino acid sequencing.
Conclusions: Our results suggest that FIB and matrix metalloproteinases secreted from bSMCs might play major roles in bSMC migration in the process of airway remodeling.

KEY WORDS:
airway remodeling, cell migration, fibronectin, matrix metalloproteinases, smooth muscle cells

ABBREVIATIONS:
BSA, Bovine serum albumin; bSMC, bronchial smooth muscle cell; CBB, Coomassie brilliant blue; DMEM, Dulbecco's modified Eagle medium; ECM, extracellular matrix; FBS, fetal bovine serum; FIB, fibronectin; HBSS, Hanks' balanced salt solution; hEGF, human epidermal growth factor; hFGF-B, human fibroblast growth factor-basic; HPF, high power field; MMP, matrix metalloproteinase; MT-MMP, membrane-type matrix metalloproteinase; PBS, phosphate buffered saline; PBST, PBS containing 0.1% Tween 20; PMSF, phenylmethylsulfonyl fluoride; SDS, sodium dodecyl sulfate; SMC, smooth muscle cell; TIMP, tissue inhibitor of metalloproteinases; uPA, urokinase plasminogen activator.

Received: 15 October 2009.
Accepted: 17 February 2010.

Allergology International 2010; 3: In Press

[Full Text PDF (503k)]