Larry W. Tsai

Cellular kinetics and modeling of bronchioalveolar stem cell response during lung regeneration.

Organ regeneration in mammals is hypothesized to require a functional pool of stem or progenitor cells, but the role of these cells in lung regeneration is unknown. Whereas postnatal regeneration of alveolar tissue has been attributed to type II alveolar epithelial cells (AECII), we reasoned that bronchioalveolar stem cells (BASCs) have the potential to contribute substantially to this process. To test this hypothesis, unilateral pneumonectomy (PNX) was performed on adult female C57/BL6 mice to stimulate compensatory lung regrowth. The density of BASCs and AECII, and morphometric and physiological measurements, were recorded on days 1, 3, 7, 14, 28, and 45 after surgery. Vital capacity was restored by day 7 after PNX. BASC numbers increased by day 3, peaked to 220% of controls (P<0.05) by day 14, and then returned to baseline after active lung regrowth was complete, whereas AECII cell densities increased to 124% of baseline (N/S). Proliferation studies revealed significant BrdU uptake in BASCs and AECII within the first 7 days after PNX. Quantitative analysis using a systems biology model was used to evaluate the potential contribution of BASCs and AECII. The model demonstrated that BASC proliferation and differentiation contributes between 0 and 25% of compensatory alveolar epithelial (type I and II cell) regrowth, demonstrating that regeneration requires a substantial contribution from AECII. The observed cell kinetic profiles can be reconciled using a dual-compartment (BASC and AECII) proliferation model assuming a linear hierarchy of BASCs, AECII, and AECI cells to achieve lung regrowth.

Epithelial cell PPARγ contributes to normal lung maturation

Peroxisome proliferator‐activated receptor (PPAR)‐γ is a member of the nuclear hormone receptor superfamily that can promote cellular differentiation and organ development. PPARγ expression has been reported in a number of pulmonary cell types, including inflammatory, mesenchymal, and epithelial cells. We find that PPARγ is prominently expressed in the airway epithelium in the mouse lung. In an effort to define the physiological role of PPARγ within the lung, we have ablated PPARγ using a novel line of mice capable of specifically targeting the airway epithelium. Airway epithelial cell PPARγ‐targeted mice display enlarged airspaces resulting from insufficient postnatal lung maturation. The increase in airspace size is accompanied by alterations in lung physiology, including increased lung volumes and decreased tissue resistance. Genome‐wide expression profiling reveals a reduction in structural extracellular matrix (ECM) gene expression in conditionally targeted mice, suggesting a disruption in epithelial‐mesenchymal interactions necessary for the establishment of normal lung structure. Expression profiling of airway epithelial cells isolated from conditionally targeted mice indicates PPARγ regulates genes encoding known PPARγ targets, additional lipid metabolism enzymes, and markers of cellular differentiation. These data reveal airway epithelial cell PPARγ is necessary for normal lung structure and function.—Simon, D. M., Arikan, M. C., Srisuma, S., Bhattacharya, S., Tsai, L. W., Ingenito, E. P., Gonzalez, F., Shapiro, S. D., and Mariani, T. J. Epithelial cell PPARγ contributes to normal lung maturation. FASEB J. 20, E710–E720 (2006)

Comparison of variable and conventional ventilation in a sheep saline lavage lung injury model.

Objective:There has recently been considerable interest in alternative lung-protective ventilation strategies such as variable ventilation (VV). We aimed at testing VV in a large animal lung injury model and exploring the mechanism of improvement in gas exchange seen with VV. Design:Randomized, controlled comparative ventilation study. Setting:Research laboratory at a veterinary hospital. Subjects:Female sheep weighing 59.8 ± 10.57 kg and excised calf lungs. Interventions:In a sheep saline lavage model of lung injury, we applied VV, whereby tidal volume (VT) and frequency (f) varied on each breath. Sheep were randomized into one of two groups (VV, n = 7; or control, n = 6) and ventilated for 4 hrs with all mean ventilation settings matched. Measurements and Main Results:Gas exchange, lung mechanics, and hemodynamic measures were recorded over the 4 hrs. VV sheep showed improvement in gas exchange (i.e., oxygenation and carbon dioxide elimination) and ventilation pressures (i.e., reduced mean and peak airway pressures) but control sheep did not. VV sheep also displayed lower-lung elastance and mechanical heterogeneity in comparison with control sheep from 2 to 4 hrs of ventilation. To study the mechanism behind improvements seen with VV, we examined the time course associated with the enhanced recruitment occurring during VV in eight saline-lavaged excised calf lungs. We found that the recruitment associated with a larger VT during VV lasted over 200 secs, nearly an order of magnitude greater than the average time interval between large VT deliveries during VV. Conclusions:The application of VV in a large animal model of lung injury results in improved gas exchange and superior lung mechanics in comparison with CV that can be explained at least partially by the long-lasting effects of the recruitments occurring during VV.

Epithelial Cell Apoptosis Causes Acute Lung Injury Masquerading as Emphysema

Theories of emphysema traditionally revolved around proteolytic destruction of extracellular matrix. Models have recently been developed that show airspace enlargement with the induction of pulmonary cell apoptosis. The purpose of this study was to determine the mechanism by which a model of epithelial cell apoptosis caused airspace enlargement. Mice were treated with either intratracheal microcystin (MC) to induce apoptosis, intratracheal porcine pancreatic elastase (PPE), or their respective vehicles. Mice from all groups were inflated and morphometry was measured at various time points. Physiology measurements were performed for airway resistance, tissue elastance, and lung volumes. The groups were further analyzed by air-saline quasistatic measurements, surfactant staining, and surfactant functional studies. Mice treated with MC showed evidence of reversible airspace enlargement. In contrast, PPE-treated mice showed irreversible airspace enlargement. The airspace enlargement in MC-treated mice was associated with an increase in elastic recoil due to an increase in alveolar surface tension. PPE-treated mice showed a loss of lung elastic recoil and normal alveolar surface tension, a pattern more consistent with human emphysema. Airspace enlargement that occurs with the MC model of pulmonary epithelial cell apoptosis displays physiology distinct from human emphysema. Reversibility, restrictive physiology due to changes in surface tension, and alveolar enlargement associated with heterogeneous alveolar collapse are most consistent with a mild acute lung injury. Inflation near total lung capacity gives the appearance of enlarged alveoli as neighboring collapsed alveoli exert tethering forces.

Rapid stimulatory effects of testosterone upon myotubule metabolism and sugar transport, as assessed by silicon microphysiometry

A considerable number of studies bave revealed bebavioral circumstances tbat give rise to small or transient differences in circulating testosterone concentrations; however, carefut consideration of androgen physiology leads to tbe disquieting conclusion that these differences are often untikely to have much physiotogic or behavioral significance. In the present report, we observe that small transients of testosterone secretion could have very rapid anabolic effects on a cultured muscle-derived cell line. Specifically, we have examined the effects of testosterone on metabolism in cultured C2CI2 myotubules, using a silicon microphysiometer. The instrument monitors cellular extrusion of protons and acidic metabolites, and such extrusion is directly linked to ATP hydrolysis, thus providing a real-time measure of cellular metabolism. Te.stosterone caused a small but significant increase in metabolism. Tbe most striking feature of this effect wa.s its rapidity, in that it occurred within 3 br. This rapid enhancement of metabolism suggested that testosterone might be enhancing substrate uptake even more rapidly. Indeed., we found that testosterone increased 2-deoxyglucose uptake within I min. The rapidity of this effect seemed to preclude mediation by classical Intracellular steroid receptors. In support of this, we were unahle to detect specific intracellular binding of testosterone. These findings sbow that testosterone can exert rapid anabolic effects on substrate transport and metabolism in myotuhules. Should this finding general to muscle in vivo, it suggests that relatively small individual differences in testosterone profiles, in response to various social interactions, may have very real consequence for subsequent muscle physiology.

Relationship between dynamic respiratory mechanics and disease heterogeneity in sheep lavage injury

Objective:Acute respiratory distress syndrome and acute lung injury are characterized by heterogeneous flooding/collapse of lung tissue. An emerging concept for managing these diseases is to set mechanical ventilation so as to minimize the impact of disease heterogeneity on lung mechanical stress and ventilation distribution. The goal of this study was to determine whether changes in lung mechanical heterogeneity with increasing positive end-expiratory pressure in an animal model of acute lung injury could be detected from the frequency responses of resistance and elastance. Design:Prospective, experimental study. Setting:Research laboratory at a veterinary hospital. Subjects:Female sheep weighing 48 ± 2 kg. Interventions:In five saline-lavaged sheep, we acquired whole-lung computed tomography scans, oxygenation, static elastance, and dynamic respiratory resistance and elastance at end-expiratory pressure levels of 7.5–20 cm H2O. Measurements and Main Results:As end-expiratory pressure increased, computed tomography-determined alveolar recruitment significantly increased but was accompanied by significant alveolar overdistension at 20 cm H2O. An optimal range of end-expiratory pressures (15–17.5 cm H2O) was identified where alveolar recruitment was significantly increased without significant overdistension. This range corresponded to the end-expiratory pressure levels that maximized oxygenation, minimized peak-to-peak ventilation pressures, and minimized indexes reflective of the mechanical heterogeneity (e.g., frequency dependence of respiratory resistance and low-frequency elastance). Static elastance did not demonstrate any significant pressure dependence or reveal an optimal end-expiratory pressure level. Conclusions:We conclude that dynamic mechanics are more sensitive than static mechanics in the assessment of the functional trade-off of recruitment relative to overdistension in a sheep model of lung injury. We anticipate that monitoring of dynamic respiratory resistance and elastance ventilator settings can be used to optimize ventilator management in acute lung injury.

A randomised trial of lung sealant versus medical therapy for advanced emphysema

Uncontrolled pilot studies demonstrated promising results of endoscopic lung volume reduction using emphysematous lung sealant (ELS) in patients with advanced, upper lobe predominant emphysema. We aimed to evaluate the safety and efficacy of ELS in a randomised controlled setting. Patients were randomised to ELS plus medical treatment or medical treatment alone. Despite early termination for business reasons and inability to assess the primary 12-month end-point, 95 out of 300 patients were successfully randomised, providing sufficient data for 3- and 6-month analysis. 57 patients (34 treatment and 23 control) had efficacy results at 3 months; 34 (21 treatment and 13 control) at 6 months. In the treatment group, 3-month lung function, dyspnoea, and quality of life improved significantly from baseline when compared to control. Improvements persisted at 6 months with >50% of treated patients experiencing clinically important improvements, including some whose lung function improved by >100%. 44% of treated patients experienced adverse events requiring hospitalisation (2.5-fold more than control, p=0.01), with two deaths in the treated cohort. Treatment responders tended to be those experiencing respiratory adverse events. Despite early termination, results show that minimally invasive ELS may be efficacious, yet significant risks (probably inflammatory) limit its current utility. Emphysematous lung sealant therapy is highly efficacious in some patients, but benefits bring significant risks http://ow.ly/JJ2vg

Matrix modulation of compensatory lung regrowth and progenitor cell proliferation in mice

Mechanical stress is an important modulator of lung morphogenesis, postnatal lung development, and compensatory lung regrowth. The effect of mechanical stress on stem or progenitor cells is unclear. We examined whether proliferative responses of epithelial progenitor cells, including dually immunoreactive (CCSP and proSP-C) progenitor cells (CCSP+/SP-C+) and type II alveolar epithelial cells (ATII), are affected by physical factors found in the lung of emphysematics, including loss of elastic recoil, reduced elastin content, and alveolar destruction. Mice underwent single lung pneumonectomy (PNY) to modulate transpulmonary pressure (mechanical stress) and to stimulate lung regeneration. Control mice underwent sham thoracotomy. Plombage of different levels was employed to partially or completely abolish this mechanical stress. Responses to graded changes in transpulmonary pressure were assessed in elastin-insufficient mice (elastin +/-, ELN+/-) and elastase-treated mice with elastase-induced emphysema. Physiological regrowth, morphometry (linear mean intercept; Lmi), and the proliferative responses of CCSP+/SP-C+, Clara cells, and ATII were evaluated. Plombage following PNY significantly reduced transpulmonary pressure, regrowth, and CCSP+/SP-C+, Clara cell, and ATII proliferation following PNY. In the ELN+/- group, CCSP+/SP-C+ and ATII proliferation responses were completely abolished, although compensatory lung regrowth was not significantly altered. In contrast, in elastase-injured mice, compensatory lung regrowth was significantly reduced, and ATII but not CCSP+/SP-C+ proliferation responses were impaired. Elastase injury also reduced the baseline abundance of CCSP+/SP-C+, and CCSP+/SP-C+ were found to be displaced from the bronchioalveolar duct junction. These data suggest that qualities of the extracellular matrix including elastin content, mechanical stress, and alveolar integrity strongly influence the regenerative capacity of the lung, and the patterns of cell proliferation in the lungs of adult mice.

Autologous Lung-Derived Mesenchymal Stem Cell Transplantation in Experimental Emphysema:

Autologous lung-derived mesenchymal stem cells (LMSCs) were transplanted endoscopically into sheep with experimental emphysema to assess their capacity to regenerate functional tissue. LMSC lines were derived from transbronchial biopsies, cloned at passage 2, expanded in culture, and labeled. A delivery scaffold containing 1% fibrinogen, 20 μg/ml of fibronectin, and 20 μg/ml of poly-L-lysine was used to promote cell attachment and spreading. Treatment animals received scaffold containing 5–10 × 106 cells/site; control animals received scaffold alone. Phenotypic markers, differentiation capacity, extracellular matrix protein expression, and paracrine function of LMSCs were characterized in vitro. Responses to LMSC transplantation in vivo were assessed in terms of clinical toxicity, lung physiology, change in tissue mass (measured by CT scanning) and perfusion (measured by scintigraphy scanning), and tissue histology. At 4-week follow-up, transplants were well tolerated and associated with increased tissue mass and lung perfusion compared to control treatment. Histology confirmed cell retention, increased cellularity, and increased extracellular matrix content following LMSC treatment. Labeled cells were distributed in the alveolar septum and peribronchiolar interstitium. Some label was also present within phagocytes, indicating that a fraction of autologous LMSCs do not survive transplantation. These results suggest that endobronchial delivery of autologous LMSCs has potential therapeutic utility for regenerating functional lung in emphysema.

Design and testing of biological scaffolds for delivering reparative cells to target sites in the lung

This study summarizes the development and testing of a scaffold to promote engraftment of cells in the distal lung. A fibrinogen–fibronectin–vitronectin hydrogel (FFVH) was developed and optimized with respect to its mechanical and biological properties for this application. In vitro, FFVH scaffolds promoted attachment, histiotypic growth and expression of basement membrane proteins by primary ovine lung mesenchymal cells derived from lung biopsies. In vivo testing was then performed to assess the ability of FFVHs to promote cell engraftment in the sheep lung. Treatment with autologous cells delivered using FFVH was clinically well tolerated. Cells labelled with a fluorescent dye (PKH‐26) were detected at treatment sites after 1 month. Tissue mass (assessed by CT imaging) and lung perfusion (assessed by nuclear scintigraphy) were increased at emphysema test sites. Post‐treatment histology demonstrated cell proliferation and increased elastin expression without scarring or collapse. No treatment‐related pathology was observed at healthy control sites. FFVH scaffolds promote cell attachment, spreading and extracellular matrix expression in vitro and apparent engraftment in vivo, with evidence of trophic effects on the surrounding tissue. Scaffolds of this type may contribute to the development of cell‐based therapies for patients with end‐stage pulmonary diseases. Copyright