Spatial and temporal changes in extracellular elastin and laminin distribution during lung alveolar development

Abstract

Lung alveolarization requires precise coordination of cell growth with extracellular matrix (ECM) synthesis and deposition. The role of extracellular matrices in alveogenesis is not fully understood, because prior knowledge is largely extrapolated from two-dimensional structural analysis. Herein, we studied temporospatial changes of two important ECM proteins, laminin and elastin that are tightly associated with alveolar capillary growth and lung elastic recoil respectively, during both mouse and human lung alveolarization. By combining protein immunofluorescence staining with two- and three-dimensional imaging, we found that the laminin network was simplified along with the thinning of septal walls during alveogenesis, and more tightly associated with alveolar endothelial cells in matured lung. In contrast, elastin fibers were initially localized to the saccular openings of nascent alveoli, forming a ring-like structure. Then, throughout alveolar growth, the number of such alveolar mouth ring-like structures increased, while the relative ring size decreased. These rings were interconnected via additional elastin fibers. The apparent patches and dots of elastin at the tips of alveolar septae found in two-dimensional images were cross sections of elastin ring fibers in the three-dimension. Thus, the previous concept that deposition of elastin at alveolar tips drives septal inward growth may potentially be conceptually challenged by our data.

Figure 1

Immunofluorescence staining of lung tissue sections of mice from P1 to P28: (A) Laminin (red)/Pecam1 (green), Pecam1 positive cells adjacent to laminin are indicated by arrows. (B) Laminin (red)/Pdpn (green). (C) Laminin staining of P7 airway (a). Pericellular basement membrane of airway smooth muscle cells and airway epithelial basement membrane are indicated by arrowhead and arrow, respectively.

Figure 2

Immunofluorescence staining of laminin (red)/Pecam1 (green) of human lung tissue sections from ages of postnatal day 1, 7 months, 3 years, 8 years, and 23 years. Right panels show magnified boxed areas highlighted in left panel.

Figure 3

Whole mount immunofluorescence staining of laminin (red) for mouse lungs of P1, P7, P10, P14, and P28. Pictures of 3-D volumetric rendering are presented with views from different angles.

Figure 4

Immunofluorescence staining of lung tissue sections of mice from P1 to P28: (A) Elastin (red)/Pecam1 (green). (B) Elastin (red)/Pdpn (green). *“finger-like” alveolar protrusions. (C) Elastin staining of P7 airway (a). Airway  smooth muscle cell layer is marked by arrows.

Figure 5

Immunofluorescence staining of elastin (red)/Pecam1 (green) of human lung tissue sections from ages of postnatal day 1, 7 months, 3 years, 8 years, and 23 years. Right panels show magnified boxed areas highlighted in left panel.

Figure 6

Whole mount immunofluorescence staining of elastin (red) for mouse lungs of P1, P7, P10, P14, and P28. Pictures of 3-D volumetric rendering are presented with views from different angles.

Figure 7

Whole mount immunofluorescence staining of elastin (red) for P7 mouse lung. (A) Stack of serial image sections of elastin staining. (B) Volumetric rendering of the serial images shown in A. DAPI counterstaining (blue) was used to display the structures of the lung tissue. “I to III” indicated three elastin rims. Arrowheads indicate the patches of elastin present at the tips of alveolar protrusions.