Hiroshi Hamakawa

Amelioration of emphysema in mice through lentiviral transduction of long-lived pulmonary alveolar macrophages

Directed gene transfer into specific cell lineages in vivo is an attractive approach for both modulating gene expression and correcting inherited mutations such as emphysema caused by human alpha1 antitrypsin (hAAT) deficiency. However, somatic tissues are mainly comprised of heterogeneous, differentiated cell lineages that can be short lived and difficult to specifically transfect. Here, we describe an intratracheally instilled lentiviral system able to deliver genes selectively to as many as 70% of alveolar macrophages (AMs) in the mouse lung. Following a single in vivo lentiviral transduction, genetically tagged AMs persisted in lung alveoli and expressed transferred genes for the lifetime of the adult mouse. A prolonged macrophage lifespan, rather than precursor cell proliferation, accounted for the surprisingly sustained presence of transduced AMs. We utilized this long-lived population to achieve localized secretion of therapeutic levels of hAAT protein in lung epithelial lining fluid. In an established mouse model of emphysema, lentivirally delivered hAAT ameliorated the progression of emphysema, as evidenced by attenuation of increased lung compliance and alveolar size. After 24 weeks of sustained gene expression, no humoral or cellular immune responses to hAAT protein were detected. Our results challenge the dogma that AMs are short lived and suggest that these differentiated cells may be a possible target cell population for in vivo gene therapy applications, including the sustained correction of hAAT deficiency.

Three-dimensional measurement of alveolar airspace volumes in normal and emphysematous lungs using micro-CT

In pulmonary emphysema, the alveolar structure progressively breaks down via a three-dimensional (3D) process that leads to airspace enlargement. The characterization of such structural changes has, however, been based on measurements from two-dimensional (2D) tissue sections or estimates of 3D structure from 2D measurements. In this study, we developed a novel silver staining method for visualizing tissue structure in 3D using micro-computed tomographic (CT) imaging, which showed that at 30 cmH20 fixing pressure, the mean alveolar airspace volume increased from 0.12 nl in normal mice to 0.44 nl and 2.14 nl in emphysematous mice, respectively, at 7 and 14 days following elastase-induced injury. We also assessed tissue structure in 2D using laser scanning confocal microscopy. The mean of the equivalent diameters of the alveolar airspaces was lower in 2D compared with 3D, while its variance was higher in 2D than in 3D in all groups. However, statistical comparisons of alveolar airspace size from normal and emphysematous mice yielded similar results in 2D and 3D: compared with control, both the mean and variance of the equivalent diameters increased by 7 days after treatment. These indexes further increased from day 7 to day 14 following treatment. During the first 7 days following treatment, the relative change in SD increased at a much faster rate compared with the relative change in mean equivalent diameter. We conclude that quantifying heterogeneity in structure can provide new insight into the pathogenesis or progression of emphysema that is enhanced by improved sensitivity using 3D measurements.

Structure–Function Relations in an Elastase-Induced Mouse Model of Emphysema

Emphysema is a progressive disease characterized by the destruction of peripheral airspaces and subsequent decline in lung function. However, the relation between structure and function during disease progression is not well understood. The objective of this study was to assess the time course of the structural, mechanical, and remodeling properties of the lung in mice after elastolytic injury. At 2, 7, and 21 days after treatment with porcine pancreatic elastase, respiratory impedance, the constituents of lung extracellular matrix, and histological sections of the lung were evaluated. In the control group, no changes were observed in the structural or functional properties, whereas, in the treatment group, the respiratory compliance and its variability significantly increased by Day 21 (P < 0.001), and the difference in parameters decreased with increasing positive end-expiratory pressure. The heterogeneity of airspace structure gradually increased over time. Conversely, the relative amounts of elastin and type I collagen exhibited a peak (P < 0.01) at Day 2, but returned to baseline levels by Day 21. Structure-function relations manifested themselves in strong correlations between compliance parameters and both mean size and heterogeneity of airspace structure (r(2) > 0.9). Similar relations were also obtained in a network model of the parenchyma in which destruction was based on the notion that mechanical forces contribute to alveolar wall rupture. We conclude that, in a mouse model of emphysema, progressive decline in lung function is sensitive to the development of airspace heterogeneity governed by local, mechanical, force-induced failure of remodeled collagen.

Combined Effects of Ventilation Mode and Positive End-Expiratory Pressure on Mechanics, Gas Exchange and the Epithelium in Mice with Acute Lung Injury

The accepted protocol to ventilate patients with acute lung injury is to use low tidal volume (VT) in combination with recruitment maneuvers or positive end-expiratory pressure (PEEP). However, an important aspect of mechanical ventilation has not been considered: the combined effects of PEEP and ventilation modes on the integrity of the epithelium. Additionally, it is implicitly assumed that the best PEEP-VT combination also protects the epithelium. We aimed to investigate the effects of ventilation mode and PEEP on respiratory mechanics, peak airway pressures and gas exchange as well as on lung surfactant and epithelial cell integrity in mice with acute lung injury. HCl-injured mice were ventilated at PEEPs of 3 and 6 cmH2O with conventional ventilation (CV), CV with intermittent large breaths (CVLB) to promote recruitment, and a new mode, variable ventilation, optimized for mice (VVN). Mechanics and gas exchange were measured during ventilation and surfactant protein (SP)-B, proSP-B and E-cadherin levels were determined from lavage and lung homogenate. PEEP had a significant effect on mechanics, gas exchange and the epithelium. The higher PEEP reduced lung collapse and improved mechanics and gas exchange but it also down regulated surfactant release and production and increased epithelial cell injury. While CVLB was better than CV, VVN outperformed CVLB in recruitment, reduced epithelial injury and, via a dynamic mechanotransduction, it also triggered increased release and production of surfactant. For long-term outcome, selection of optimal PEEP and ventilation mode may be based on balancing lung physiology with epithelial injury.

Scale dependence of structure-function relationship in the emphysematous mouse lung

The purpose of this study was to determine how the initial distribution of elastase in mouse lungs determines the time course of tissue destruction and how structural heterogeneity at different spatial scales influences lung function. We evaluated lung function and alveolar structure in normal and emphysematous C57BL/6 mice at 2 and 21 days following orotracheal treatment with porcine pancreatic elastase (PPE). Initial distribution of elastase 1 h after treatment was assessed using red fluorescently labeled PPE (f-PPE) by laser scanning confocal microscopy. From measured input impedance of the respiratory system, the global lung compliance, and the variability of regional compliance were obtained. Lungs were fixed and equivalent airspace diameters were measured in four lobes of the right lung and three regions of the left lung. At day 2 and day 21, the mean airspace diameter of each region was significantly enlarged which was accompanied by an increased inter-regional heterogeneity. The deposition of f-PPE on day 0 was much more heterogeneous than the inter-regional diameters at both day 2 and day 21 and, at day 21, this reached statistical significance (p < 0.05). Microscale heterogeneity characterized by the overall variability of airspace diameters correlated significantly better with compliance than macroscale or inter-regional heterogeneity. Furthermore, while the spatial distribution of the inflammatory response does not seem to follow that of the elastase deposition, it correlates with the strongest regional determinant of lung function. These results may help interpret lung function decline in terms of structural deterioration in human patients with emphysema.