Micromechanical forces regulate vascular patterning in the chick chorioallantoic membrane (15.3)

Glycoconjugate expression in the extracellular matrix of the mouse lung (87.7)

Structural Remodeling of the Post‐pneumonectomy Lung is Characterized by Septal Retraction and Alveolar Duct Dilation

Murine pneumonectomy (PNX) is an established model of adult compensatory lung growth, involving structural remodeling of the peripheral parenchyma. Following left PNX, positron emission tomography and computerized tomography have demonstrated heterogeneous growth patterns within the remaining right lung. To characterize the structural changes associated with these observations, we analyzed histological sections of the right lung between 3 and 22 days after PNX. Within 3 days of PNX, alveolar septae were significantly shorter (p<0.05) in a majority of alveolar ducts, while septal angle remained unchanged. Septal retraction resulted in an apparent dilation of the alveolar duct with a signific…

Elastin Cables Define the Axial Connective Tissue System in the Murine Lung

The axial connective tissue system is a fiber continuum of the lung that maintains alveolar surface area during changes in lung volume. Although the molecular anatomy of the axial system remains undefined, the fiber continuum of the lung is central to contemporary models of lung micromechanics and alveolar regeneration. To provide a detailed molecular structure of the axial connective tissue system, we examined the extracellular matrix of murine lungs. The lungs were decellularized using a 24 hr detergent treatment protocol. Systematic evaluation of the decellularized lungs demonstrated no residual cellular debris; morphometry demonstrated a mean 39 ± 7% reduction in lung dimensions. Scanni…

Evidence for pleural epithelial-mesenchymal transition in murine compensatory lung growth

In many mammals, including rodents and humans, removal of one lung results in the compensatory growth of the remaining lung; however, the mechanism of compensatory lung growth is unknown. Here, we investigated the changes in morphology and phenotype of pleural cells after pneumonectomy. Between days 1 and 3 after pneumonectomy, cells expressing α-smooth muscle actin (SMA), a cytoplasmic marker of myofibroblasts, were significantly increased in the pleura compared to surgical controls (p < .01). Scanning electron microscopy of the pleural surface 3 days post-pneumonectomy demonstrated regions of the pleura with morphologic features consistent with epithelial-mesenchymal transition (EMT); nam…

Single-Cell Transcriptional Profiling of Cells Derived From Regenerating Alveolar Ducts

Lung regeneration occurs in a variety of adult mammals after surgical removal of one lung (pneumonectomy). Previous studies of murine post-pneumonectomy lung growth have identified regenerative “hotspots” in subpleural alveolar ducts; however, the cell-types participating in this process remain unclear. To identify the single cells participating in post-pneumonectomy lung growth, we used laser microdissection, enzymatic digestion and microfluidic isolation. Single-cell transcriptional analysis of the murine alveolar duct cells was performed using the C1 integrated fluidic circuit (Fluidigm) and a custom PCR panel designed for lung growth and repair genes. The multi-dimensional data set was …

Laser Microdissection of the Alveolar Duct Enables Single-Cell Genomic Analysis

Complex tissues such as the lung are composed of structural hierarchies such as alveoli, alveolar ducts, and lobules. Some structural units, such as the alveolar duct, appear to participate in tissue repair as well as the development of bronchioalveolar carcinoma. Here, we demonstrate an approach to conduct laser microdissection of the lung alveolar duct for single-cell PCR analysis. Our approach involved three steps. The initial preparation used mechanical sectioning of the lung tissue with sufficient thickness to encompass the structure of interest. In the case of the alveolar duct, the precision-cut lung slices were 200µm thick; the slices were processed using near-physiologic conditions…

Sequence of vascular patterning and gene transcription in the chick chorioallantoic membrane (15.1)

Introduction: The chick chorioallantoic membrane (CAM) is a well-established model of both vasculogenesis and angiogenesis; however, little is known about the genetic control of vascular patterning in the CAM. Methods: Using recent advances in chicken genomics, we investigated the relative expression of 84 angiogenesis genes during the growth and remodeling of the CAM microcirculatory network. Chick embryos, cultured ex ovo, were studied during embryonic development days (EDD) 8-14. UV laser microdissection was used to harvest capillary plexus and 1st, 2nd, and 3rd order conducting vessels for qRT-PCR analysis. Results: Two transcription peaks were observed between EDD 8 and 14. The first p…

Post‐pneumonectomy Lung Deformation is Associated with Alveolar Type II Cell Apoptosis and Altered Parenchymal Mechanics

Remodeling of alveolar septa after murine pneumonectomy

In most mammals, removing one lung (pneumonectomy) results in the compensatory growth of the remaining lung. In mice, stereological observations have demonstrated an increase in the number of mature alveoli; however, anatomic evidence of the early phases of alveolar growth has remained elusive. To identify changes in the lung microstructure associated with neoalveolarization, we used tissue histology, electron microscopy, and synchrotron imaging to examine the configuration of the alveolar duct after murine pneumonectomy. Systematic histological examination of the cardiac lobe demonstrated no change in the relative frequency of dihedral angle components (Ends, Bends, and Junctions) ( P &gt…

Stretch-induced Intussuceptive and Sprouting Angiogenesis in the Chick Chorioallantoic Membrane

Vascular systems grow and remodel in response to not only metabolic needs, but also mechanical influences as well. Here, we investigated the influence of tissue-level mechanical forces on the patterning and structure of the chick chorioallantoic membrane (CAM) microcirculation. A dipole stretch field was applied to the CAM using custom computer-controlled servomotors. The topography of the stretch field was mapped using finite element models. After 3 days of stretch, Sholl analysis of the CAM demonstrated a 7-fold increase in conducting vessel intersections within the stretch field (p 0.05). In contrast, corrosion casting and SEM of the stretch field capillary meshwork demonstrated intense …

Deformation-induced transitional myofibroblasts contribute to compensatory lung growth

In many mammals, including humans, removal of one lung (pneumonectomy) results in the compensatory growth of the remaining lung. Compensatory growth involves not only an increase in lung size, but also an increase in the number of alveoli in the peripheral lung; however, the process of compensatory neoalveolarization remains poorly understood. Here, we show that the expression of α-smooth muscle actin (SMA)—a cytoplasmic protein characteristic of myofibroblasts—is induced in the pleura following pneumonectomy. SMA induction appears to be dependent on pleural deformation (stretch) as induction is prevented by plombage or phrenic nerve transection (P < 0.001). Within 3 days of pneumonectomy, …

Extracellular Assembly of the Elastin Cable Line Element in the Developing Lung

In the normal lung, a dominant structural element is an elastic "line element" that originates in the central bronchi and inserts into the distal airspaces. Despite its structural importance, the process that leads to development of the cable line element is unknown. To investigate the morphologic events contributing to its development, we used optical clearing methods to examine the postnatal rat lung. An unexpected finding was numerous spheres, with a median diameter of 1-2 µm, within the primary septa of the rat lung. The spheres demonstrated green autofluorescence, selective fluorescent eosin staining, reactivity with carboxyfluorescein succinimidyl ester, and specific labeling with ant…