Recellularization of whole decellularized lung scaffolds provides a novel approach for generating functional lung tissue ex vivo for subsequent clinical transplantation. To explore the potential utility of stem and progenitor cells in this model, we investigated recellularization of decellularized whole mouse lungs after intratracheal inoculation of bone marrow-derived mesenchymal stromal cells (MSCs). The decellularized lungs maintained structural features of native lungs, including intact vasculature, ability to undergo ventilation, and an extracellular matrix (ECM) scaffold consisting primarily of collagens I and IV, laminin, and fibronectin. However, even in the absence of intact cells or nuclei, a number of cell-associated (non-ECM) proteins were detected using mass spectroscopy, western blots, and immunohistochemistry. MSCs initially homed and engrafted to regions enriched in types I and IV collagen, laminin, and fibronectin, and subsequently proliferated and migrated toward regions enriched in types I and IV collagen and laminin but not provisional matrix (fibronectin). MSCs cultured for up to 1 month in either basal MSC medium or in a small airways growth media (SAGM) localized in both parenchymal and airway regions and demonstrated several different morphologies. However, while MSCs cultured in basal medium increased in number, MSCs cultured in SAGM decreased in number over 1 month. Under both media conditions, the MSCs predominantly expressed genes consistent with mesenchymal and osteoblast phenotype. Despite a transient expression of the lung precursor TTF-1, no other airway or alveolar genes or vascular genes were expressed. These studies highlight the power of whole decellularized lung scaffolds to study functional recellularization with MSCs and other cells.

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Inappropriate mechanical ventilation in patients with acute respiratory distress syndrome can lead to ventilator-induced lung injury (VILI) and increase the morbidity and mortality. Reopening collapsed lung units may significantly reduce VILI, but the mechanisms governing lung recruitment are unclear. We thus investigated the dynamics of lung recruitment at the alveolar level. Rats (n = 6) were anesthetized and mechanically ventilated. The lungs were then lavaged with saline to simulate acute respiratory distress syndrome (ARDS). A left thoracotomy was performed, and an in vivo microscope was placed on the lung surface. The lung was recruited to three recruitment pressures (RP) of 20, 30, or 40 cmH(2)O for 40 s while subpleural alveoli were continuously filmed. Following measurement of microscopic alveolar recruitment, the lungs were excised, and macroscopic gross lung recruitment was digitally filmed. Recruitment was quantified by computer image analysis, and data were interpreted using a mathematical model. The majority of alveolar recruitment (78.3 +/- 7.4 and 84.6 +/- 5.1%) occurred in the first 2 s (T2) following application of RP 30 and 40, respectively. Only 51.9 +/- 5.4% of the microscopic field was recruited by T2 with RP 20. There was limited recruitment from T2 to T40 at all RPs. The majority of gross lung recruitment also occurred by T2 with gradual recruitment to T40. The data were accurately predicted by a mathematical model incorporating the effects of both pressure and time. Alveolar recruitment is determined by the magnitude of recruiting pressure and length of time pressure is applied, a concept supported by our mathematical model. Such a temporal dependence of alveolar recruitment needs to be considered when recruitment maneuvers for clinical application are designed.

Rationale: Nuclear factor (NF)-kB is a prominent proinflammatory transcription factor that plays a critical role in allergic airway disease. Previous studies demonstrated that inhibition of NF-kB in airway epithelium causes attenuation of allergic inflammation. Objectives: We sought to determine if selective activation of NF-kB within the airway epithelium in the absence of other agonists is sufficient to cause allergic airway disease. Methods: A transgenic mouse expressing a doxycycline (Dox)-inducible, constitutively active (CA) version of inhibitor of kB (IkB) kinase-b (IKKb) under transcriptional control of the rat CC10 promoter, was generated. Measurements and Main Results: After administration of Dox, expression of the CA-IKKb transgene induced the nuclear translocation of RelA in airway epithelium. IKKb-triggered activation of NF-kB led to an increased content of neutrophils and lymphocytes, and concomitant production of proinflammatory mediators, responses that were not observed in transgenic mice not receiving Dox, or in transgenenegative littermate control animals fed Dox. Unexpectedly, expression of the IKKb transgene in airway epithelium was sufficient to cause airway hyperresponsiveness and smooth muscle thickening in absence of an antigen sensitization and challenge regimen, the presence of eosinophils, or the induction of mucus metaplasia.Conclusions: These findings demonstrate that selective activation NFkB in airway epithelium is sufficient to induce airway hyperresponsiveness and smooth muscle thickening, which are both critical features of allergic airway disease.

Rationale: Allergically inflamed mice exhibit airway hyperresponsiveness to inhaled methacholine, which computer simulations of lung impedance suggest is due to enhanced lung derecruitment and which we sought to verify in the present study. Methods: BALB/c mice were sensitized and challenged with ovalbumin to induce allergic inflammation; the control mice were sensitized but received no challenge. The mice were then challenged with inhaled methacholine and respiratory system impedance tracked for the following 10 minutes. Respiratory elastance (H ) was estimated from each impedance measurement. One group of mice was ventilated with 100% O 2 during this procedure and another group was ventilated with air. After the procedure, the mice were killed and ventilated with pure N 2 , after which the trachea was tied off and the lungs were imaged with micro-computed tomography (micro-CT).Results: H was significantly higher in allergic mice than in control animals after methacholine challenge. The ratio of H at the end of the measurement period between allergic and nonallergic mice ventilated with O 2 was 1.36, indicating substantial derecruitment in the allergic animals. The ratio between lung volumes determined by micro-CT in the control and the allergic mice was also 1.36, indicative of a corresponding volume loss due to absorption atelectasis. Micro-CT images and histograms of Hounsfield units from the lungs also showed increased volume loss in the allergic mice compared with control animals after methacholine challenge. Conclusions: These results support the conclusion that airway closure is a major component of hyperresponsiveness in allergically inflamed mice.Keywords: asthma; micro-computed tomography; input impedance; lung derecruitment; lung volume Different mechanisms can lead to airway hyperresponsiveness (AHR) in animal models, not all of which have equal relevance to human asthma. It is therefore crucial to elucidate the mechanistic basis of AHR in any given animal model of asthma to understand its relationship to the human condition. We recently reported that the response of acutely allergically inflamed BALB/c mice to challenge with methacholine aerosol is likely due entirely to enhanced closure of peripheral airways secondary to increased (Received in original form October 3, 2006; accepted in final form January 25, 2007 ) Supported by NIH grants R01 HL67273 and NCRR-COBRE P20 RR15557.Correspondence and requests for reprints should be addressed to Lennart K. AT A GLANCE COMMENTARY Scientific Knowledge on the SubjectAirway hyperresponsiveness in mouse models of asthma has traditionally been attributed to contraction of airway smooth muscle. Recent studies suggest, however, that airway closure may play a more significant role. What This Study Adds to the FieldUsing the forced oscillation technique and micro-computed tomography, we now show that airway hyperresponsiveness in allergic mice can be largely explained by airway closure.airway mucosal thickness and secretions (1). This stands in marked contras...