Alteration of cystic airway mesenchyme in congenital pulmonary airway malformation

Abstract

Congenital pulmonary airway malformation (CPAM) is the most common congenital lesion detected in the neonatal lung, which may lead to respiratory distress, infection, and pneumothorax. CPAM is thought to result from abnormal branching morphogenesis during fetal lung development, arising from different locations within the developing respiratory tract. However, the pathogenic mechanisms are unknown, and previous studies have focused on abnormalities in airway epithelial cells. We have analyzed 13 excised lung specimens from infants (age < 1 year) with a confirmed diagnosis of type 2 CPAM, which is supposed to be derived from abnormal growth of intrapulmonary distal airways. By examining the mesenchymal components including smooth muscle cells, laminin, and elastin in airway and cystic walls using immunofluorescence staining, we found that the thickness and area of the smooth muscle layer underlining the airway cysts in these CPAM tissue sections were significantly decreased compared with those in bronchiolar walls of normal controls. Extracellular elastin fibers were also visually reduced or absent in airway cystic walls. In particular, a layer of elastin fibers seen in normal lung between airway epithelia and underlying smooth muscle cells was missing in type 2 CPAM samples. Thus, our data demonstrate for the first time that airway cystic lesions in type 2 CPAM occur not only in airway epithelial cells, but also in adjacent mesenchymal tissues, including airway smooth muscle cells and their extracellular protein products. This provides a new direction to study the molecular and cellular mechanisms of CPAM pathogenesis in human.

Figure 1

Validation of type 2 CPAM histopathology. Cystic airway structures from three randomly selected CPAM lung specimens (1–3) are shown by H&E staining. Back-to-back bronchiole-like structures are shown by the asterisks. Normal distal bronchiolar structure from a control lung was included for comparison.

Figure 2

Reduced smooth muscle layers in cystic airway walls of CPAM. Compared to normal bronchiolar walls, the thickness of the smooth muscle cell layer, detected by MYH11 staining (red), is reduced in CPAM samples. Ciliated epithelial cells were co-stained by anti-TUBB4A antibody (green), and cell nuclei were counterstained with DAPI (blue). Images from 3 randomly selected type 2 CPAM specimens and 3 normal lungs are presented.

Figure 3

Semi-quantitative measurements and comparisons of airway smooth muscle layers. (A) Schematic illustration for average smooth muscle (SM, red) thickness and SM area. The mucous epithelial layer is marked as green. The mucous membrane length (L, shown as a double arrow line) was used for normalization. (B) Sum smooth muscle area normalized by mucous membrane length shown as a dot plot, with the bars indicating mean ± se. (C) Average smooth muscle layer thickness measurement shown as a dot plot, with mean ± se. (B,C) Comparisons were performed between cystic airways of type 2 CPAM samples (n = 13) and normal bronchioles in control lungs (Ctrl, n = 7) using non-parametric Mann-Whitney U test. *P < 0.05.

Figure 4

Changes of extracellular matrix proteins in cystic airways of type 2 CPAM. (A) Laminin (green) was co-immunostained with vascular endothelial marker PECAM1 (red) and presented in separate panels. (B) Elastin (green) was co-immunostained with smooth muscle cell marker ACTA2 (red). Cell nuclei were counterstained with DAPI (blue). The lower panel shows a magnified small area from upper panel. Arrow: An elastin layer between epithelia and SMCs. Control samples were from normal age-matched lungs, and type 2 CPAM samples (CPAM-1 or -2) were from different patients.