Robert D. Bennett

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. Scanning electron microscopy (SEM) demonstrated an intact structural hierarchy within the decellularized lung. Light, fluorescence, and SEM of precision‐cut lung slices demonstrated that alveolar duct structure was defined by a cable line element encased in basement membrane. The cable line element arose in the distal airways, passed through septal tips and inserted into neighboring blood vessels and visceral pleura. The ropelike appearance, collagenase resistance and anti‐elastin immunostaining indicated that the cable was an elastin macromolecule. Our results indicate that the helical line element of the axial connective tissue system is composed of an elastin cable that not only defines the structure of the alveolar duct, but also integrates the axial connective tissue system into visceral pleura and peripheral blood vessels. Anat Rec, 298:1960–1968, 2015.

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. (1) 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 200 μm thick; the slices were processed using near-physiologic conditions to preserve the state of viable cells. (2) The lung slices were examined by transmission light microscopy to target the alveolar duct. The air-filled lung was sufficiently accessible by light microscopy that counterstains or fluorescent labels were unnecessary to identify the alveolar duct. (3) The enzymatic and microfluidic isolation of single cells allowed for the harvest of as few as several thousand cells for PCR analysis. Microfluidics based arrays were used to measure the expression of selected marker genes in individual cells to characterize different cell populations. Preliminary work suggests the unique value of this approach to understand the intra- and intercellular interactions within the regenerating alveolar duct.

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 3days of stretch, Sholl analysis of the CAM demonstrated a 7-fold increase in conducting vessel intersections within the stretch field (p<0.01). The morphometric analysis of intravital microscopy and scanning electron microscopy (SEM) images demonstrated that the increase vessel density was a result of an increase in interbranch distance (p<0.01) and a decrease in bifurcation angles (p<0.01); there was no significant increase in conducting vessel number (p>0.05). In contrast, corrosion casting and SEM of the stretch field capillary meshwork demonstrated intense sprouting and intussusceptive angiogenesis. Both planar surface area (p<0.05) and pillar density (p<0.01) were significantly increased relative to control regions of the CAM. We conclude that a uniaxial stretch field stimulates the axial growth and realignment of conducting vessels as well as intussusceptive and sprouting angiogenesis within the gas exchange capillaries of the ex ovo CAM.

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, the frequency of SMA+ cells in subpleural alveolar ducts was significantly increased (P < 0.01). To determine the functional activity of these SMA+ cells, we isolated regenerating alveolar ducts by laser microdissection and analyzed individual cells using microfluidic single-cell quantitative PCR. Single cells expressing the SMA (Acta2) gene demonstrated significantly greater transcriptional activity than endothelial cells or other discrete cell populations in the alveolar duct (P < 0.05). The transcriptional activity of the Acta2+ cells, including expression of TGF signaling as well as repair-related genes, suggests that these myofibroblast-like cells contribute to compensatory lung growth.

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); namely, cells with disrupted intercellular junctions and an acquired mesenchymal (rounded and fusiform) morphotype. To detect the migration of the transitional pleural cells into the lung, a biotin tracer was used to label the pleural mesothelial cells at the time of surgery. By post-operative day 3, image cytometry of post-pneumonectomy subpleural alveoli demonstrated a 40-fold increase in biotin+ cells relative to pneumonectomy-plus-plombage controls (p < .01). Suggesting a similar origin in space and time, the distribution of cells expressing biotin, SMA, or vimentin demonstrated a strong spatial autocorrelation in the subpleural lung (p < .001). We conclude that post-pneumonectomy compensatory lung growth involves EMT with the migration of transitional mesothelial cells into subpleural alveoli.

Extracellular Assembly of the Elastin Cable Line Element in the Developing Lung: ELASTIN LINE ELEMENT IN 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 anti‐tropoelastin monoclonal antibody—findings consistent with tropoelastin. The sphere number peaked on rat postnatal day 4 (P4) and were rare by P14. The disappearance of the spheres was coincident with the development of the cable line element in the rat lung. Transmission electron microscopy demonstrated no consistent association between parenchymal cells and sphere alignment. In contrast, the alignment of tropoelastin spheres appeared to be the direct result of interactions of scaffold proteins including collagen fibers and fibrillin microfibrils. We conclude that the spatial organization of the cable line element appears to be independent of tropoelastin deposition, but dependent on crosslinking to scaffold proteins within the primary septa. Anat Rec, 300:1670–1679, 2017.

Transcriptional Regulation of Post-Pneumonectomy Angiogenesis in Peripheral Lung Regeneration

RESULTS: We identified 8,930 lung resection patients; 59.5% of resections were performed for lung cancer and of these, 73% were lobectomy. Overall, 56% of cases were performed using VATS. Morbidity and mortality rates were 9% and 1.2% for VATS vs 15.3% and 2% for THOR (p<0.001). Median length of stay was 4 days for VATS (interquartile range [IQR] 2-6 days), and 5 days (IQR 4-8 days) for THOR (p<0.001). Unplanned readmission occurred within 30 days in 7.4% of patients and was not dependent on surgical approach or preoperative diagnosis. The reason for readmission was more likely related to air leak in the VATS group vs infections in the THOR group. Multivariate predictors of readmission included American Society of Anesthesiologists (ASA) 3, pneumonectomy, and the occurrence of postoperative complications as shown in the table.