Triggering the Induction of Myofibroblast and Fibrogenesis by Airway Epithelial Shedding

Abstract

Myofibroblasts have been thought to participate in subepithelial fibrosis in asthma, but the mechanism of myofibroblast induction has not been fully understood. In this study we investigated injury-related myofibroblast induction in a coculture system of guinea-pig epithelial cells and fibroblasts cocultured in a human amnion chamber. After pseudostratified epithelial cells were mechanically scraped, migrated flat epithelial cells differentiated into cuboidal appearances on Day 4 and then returned to their original shapes on Day 8. During the course of the epithelial redifferentiation, it was found by Northern blot analysis, immunohistochemistry for alpha-smooth muscle actin, and electron microscopic observation that the myofibroblasts were transiently induced on Day 4. The myofibroblast induction was inhibited by the blocking of transforming growth factor (TGF)-beta1 and thrombospondin (TSP)-1, indicating that the activation of TGF-beta1 by TSP-1 would induce myofibroblasts. This finding was also supported by a transient upregulation of TSP immunoreactivity and TSP-1 messenger RNA (mRNA) in fibroblasts. Interestingly, epithelial injury reduced TGF-beta1 immunoreactivity in the amnion membrane but did not affect TGF-beta1 mRNA in epithelial cells and fibroblasts, indicating that TGF-beta1 supplied from the extracellular matrix can participate in myofibroblast induction. Concurrently with myofibroblast induction, procollagen type I and III mRNAs were upregulated in fibroblasts, and obvious collagen deposition was observed ultrastructurally around the myofibroblasts compared with the fibroblasts. These results indicate that induced myofibroblasts can be functionally more active in producing collagen than are resting fibroblasts. The present study suggests that epithelial injury stimulates TGF-beta1 release from the extracellular matrix and its activation via TSP-1 production, causing collagen synthesis through myofibroblast induction.

Identification of Circulating Fibrocytes as Precursors of Bronchial Myofibroblasts in Asthma

“…In another set of experiments we therefore evaluated whether human circulating fibrocytes can differentiate into myofibroblast-like cells under the influence of two fibrogenic cytokines, ET-1 and TGF-␤1, that fare known to function as important myofibroblast promoters (15,19,20,43,44) and are released in exaggerated quantities in the airways of asthmatic patients (30, 31, 40 -42). We found that fibrocytes cultured for 6 days in SFM alone were able to grow and constitutively produce fibronectin and collagen III with a pattern similar to mature lung fibroblasts (44,46).…”

“…Although it has been proposed that myofibroblasts originate from airway resident cells, particularly tissue fibroblasts or myocytes (15)(16)(17)(18)(19)(20), there may be a circulating precursor still unidentified. Previous studies (21,22) have described a population of circulating cells, termed fibrocytes, that express fibroblast products as well as the hemopoietic stem cell Ag CD34, enter sites of tissue injury, and localize to areas of extracellular matrix deposition.…”

“…The expression of ␣-smooth muscle actin (␣-SMA) 3 is a marker of differentiation into myofibroblasts (17,18,20), and fibrocytes are the only cell population known to express both CD34 and ␣-SMA (21)(22)(23)29). To detect fibrocytes that had undergone local differentiation into myofibroblast-like cells, adjacent sections were therefore double labeled with the anti-CD34 mAb indicated above and with an anti ␣-SMA mAb (DAKO).…”

“…To evaluate the expression of ␣-SMA as a marker of differentiation into myofibroblasts (17,18,20), cells cultured for the indicated periods of time in the absence or the presence of either ET-1 or TGF-␤1 were resuspended and incubated with an FITC-conjugated Ab against ␣-SMA (Sigma-Aldrich). The proportion of labeled fibrocytes was estimated by flow cytometry using Coulter beads for calibration (37).…”

“…Both these cytokines are released in excess in the airways of asthmatic patients (30, 31, 40 -42), particularly during natural or induced acute exacerbations of the disease (40,41). They are important promoters of myofibroblast differentiation in vitro (15,19,20,43,44) and are considered to play a role in the remodeling response to tissue injury by inflammation in the lung (5,45).…”