Asthmatic airway epithelial cells differentially regulate fibroblast expression of extracellular matrix components

Abstract

Background: Airway remodeling might explain lung function decline among asthmatic children. Extracellular matrix (ECM) deposition by human lung fibroblasts (HLFs) is implicated in airway remodeling. Airway epithelial cell (AEC) signaling might regulate HLF ECM expression.

Objectives: We sought to determine whether AECs from asthmatic children differentially regulate HLF expression of ECM constituents.

Methods: Primary AECs were obtained from well-characterized atopic asthmatic (n = 10) and healthy (n = 10) children intubated during anesthesia for an elective surgical procedure. AECs were differentiated at an air-liquid interface for 3 weeks and then cocultured with HLFs from a healthy child for 96 hours. Collagen I (COL1A1), collagen III (COL3A1), hyaluronan synthase (HAS) 2, and fibronectin expression by HLFs and prostaglandin E2 synthase (PGE2S) expression by AECs were assessed by using RT-PCR. TGF-β1 and TGF-β2 concentrations in media were measured by using ELISA.

Results: COL1A1 and COL3A1 expression by HLFs cocultured with AECs from asthmatic patients was greater than that by HLFs cocultured with AECs from healthy subjects (2.2-fold, P < .02; 10.8-fold, P < .02). HAS2 expression by HLFs cocultured with AECs from asthmatic patients was 2.5-fold higher than that by HLFs cocultured with AECs from healthy subjects (P < .002). Fibronectin expression by HLFs cocultured with AECs from asthmatic patients was significantly greater than that by HLFs alone. TGF-β2 activity was increased in cocultures of HLFs with AECs from asthmatic patients (P < .05), whereas PGES2 was downregulated in AEC-HLF cocultures (2.2-fold, P < .006).

Conclusions: HLFs cocultured with AECs from asthmatic patients showed differential expression of the ECM constituents COL1A1 and COL3A1 and HAS2 compared with HLFs cocultured with AECs from healthy subjects. These findings support a role for altered ECM production in asthmatic airway remodeling, possibly regulated by unbalanced AEC signaling.

Keywords: Asthma; TGF-β2; airway remodeling; children; collagen I; collagen III; epithelial cells; extracellular matrix; fibronectin; human lung fibroblasts; hyaluronic acid.

Fig 1

Panel A: Expression of Collagen I (COL1A1) mRNA by HLFs alone compared to co-culture with healthy AECs or asthmatic AECs. Panel B: Expression of Collagen III (COL3A1) mRNA by HLFs alone compared to co-culture with healthy AECs or asthmatic AECs. Panel C: Expression of Hyaluronic Acid Synthase 2 (HAS2) mRNA by HLFs alone compared to co-culture with healthy AECs or asthmatic AECs. Panel D: Expression of Fibronectin (FNDC) by HLFs alone compared to co-culture with healthy AECs or asthmatic AECs.

Fig 2

Collagen III expression assessed by immunohistochemistry between HLFs alone (Panel A), HLFs+Healthy AECs (Panel B), and HLFs+Asthmatic AECs (Panel C).

Fig 3

Hyaluronic Acid (HA) expression assessed by immunohistochemistry between HLFs alone (Panel A), HLFs+Healthy AECs (Panel B), and HLFs+Asthmatic AECs (Panel C).

Fig 4

Concentrations of TGFb1 measured in culture media collected at 96 hours from HLFs alone compared to healthy AEC+HLF co-cultures, and asthmatic AEC-HLF co-cultures (Panel A; lines at means with SD). Concentrations of TGFb2 measured in culture media collected at the 96 hour time point from HLF alone, HLF+healthy AEC, and HLF+asthmatic AEC co-cultures (Panel B; lines at medians with IQR).

Fig 5

Prostaglandin E2 synthase (PGE2S) mRNA expression is depicted on the left side of the figure in either asthmatic airway epithelial cells (AEC) or healthy AEC. The right side of the figure demonstrates mRNA expression of prostaglandin-endoperoxide synthase 2 (PTGS2/COX-2) in asthmatic or healthy AEC, respectively.