No abstract

Mechanical ventilation generates injurious forces that exacerbate lung injury. These forces disrupt lung barrier integrity, trigger proinflammatory mediator release, and differentially regulate genes and non-coding oligonucleotides including microRNAs. In this study, we identify miR-146a as a mechanosensitive microRNA in alveolar macrophages that has therapeutic potential to mitigate lung injury during mechanical ventilation. We use humanized in-vitro systems, mouse models, and biospecimens from patients to elucidate the expression dynamics of miR-146a needed to decrease lung injury during mechanical ventilation. We find that the endogenous increase in miR-146a following injurious ventilation is not sufficient to prevent lung injury. However, when miR-146a is highly overexpressed using a nanoparticle delivery platform it is sufficient to prevent injury. These data indicate that the endogenous increase in microRNA-146a during mechanical ventilation is a compensatory response that partially limits injury and that nanoparticle delivery of miR-146a is an effective strategy for mitigating lung injury during mechanical ventilation.

Risk factors of heart valve disease are well defined and prolonged exposure throughout life leads to degeneration and dysfunction in up to 33% of the population. While aortic valve replacement remains the most common need for cardiovascular surgery particularly in those aged over 65, the underlying mechanisms of progressive deterioration are unknown. In other cardiovascular systems, a decline in endothelial cell integrity and function play a major role in promoting pathological changes, and while similar mechanisms have been speculated in the valves, studies to support this are lacking. The goal of this study was to examine age-related changes in valve endothelial cell (VEC) distribution, morphology, function and transcriptomes during critical stages of valve development (embryonic), growth (post natal (PN)), maintenance (young adult) and aging (aging adult). Using a combination of in vivo mouse, and in vitro porcine assays we show that VEC function including, nitric oxide bioavailability, metabolism, endothelial-to-mesenchymal potential, membrane self repair and proliferation decline with age. In addition, density of VEC distribution along the endothelium decreases and this is associated with changes in morphology, decreased cell-cell interactions, and increased permeability. These changes are supported by RNA-seq analysis showing that focal adhesion-, cell cycle-, and oxidative phosphorylation-associated biological processes are negatively impacted by aging. Furthermore, by performing high-throughput analysis we are able to report the differential and common transcriptomes of VECs at each time point that can provide insights into the mechanisms underlying age-related dysfunction. These studies suggest that maturation of heart valves over time is a multifactorial process and this study has identified several key parameters that may contribute to impairment of the valve to maintain critical structure-function relationships; leading to degeneration and disease.

Ex vivo lung perfusion (EVLP) is a powerful experimental model for isolated lung research. EVLP allows for the lungs to be manipulated and characterized in an external environment so that the effect of specific ventilation/perfusion variables can be studied independent of other confounding physiologic contributions. At the same time, EVLP allows for normal organ level function and real-time monitoring of pulmonary physiology and mechanics. As a result, this technique provides unique advantages over in vivo and in vitro models. Small and large animal models of EVLP have been developed and each of these models has their strengths and weaknesses. In this manuscript, we provide insight into the relative strengths of each model and describe how the development of advanced EVLP protocols is leading to a novel experimental platform that can be used to answer critical questions in pulmonary physiology and transplant medicine.

The number of acceptable donor lungs available for lung transplantation is severely limited due to poor quality. Ex-Vivo Lung Perfusion (EVLP) has allowed lung transplantation in humans to become more readily available by enabling the ability to assess organs and expand the donor pool. As this technology expands and improves, the ability to potentially evaluate and improve the quality of substandard lungs prior to transplant is a critical need. In order to more rigorously evaluate these approaches, a reproducible animal model needs to be established that would allow for testing of improved techniques and management of the donated lungs as well as to the lung-transplant recipient. In addition, an EVLP animal model of associated pathologies, e.g., ventilation induced lung injury (VILI), would provide a novel method to evaluate treatments for these pathologies. Here, we describe the development of a rat EVLP lung program and refinements to this method that allow for a reproducible model for future expansion. We also describe the application of this EVLP system to model VILI in rat lungs. The goal is to provide the research community with key information and "pearls of wisdom"/techniques that arose from trial and error and are critical to establishing an EVLP system that is robust and reproducible.

We have examined the interactions between polymer-coated anionic (Ag-COOH) and cationic (Ag-NH) silver nanoparticles, and net-anionic lipid monolayers using dynamic surface pressure measurements. Monolayers composed of saturated or monounsaturated mixtures of anionic phosphatidylglycerol (PG) and zwitterionic phosphatidylcholine (PC) lipids (3:1 molar ratio) were used to determine how lipid packing and monolayer phase state influence the extent of nanoparticle binding and the monolayer response. Anionic Ag-COOH inserted into saturated dipalmitoyl-PC/PG (DPPC/DPPG) and dioleoyl-PC/PG (DOPC/DOPG) monolayers at a low initial surface pressure (10 mN m) and caused lipid condensation at high initial surface pressures (20 and 30 mN m). Hydrophobic interactions were responsible for insertion, while electrostatic and charge-dipole interactions with PCs were responsible for condensation. In contrast, cationic Ag-NH inserted only into saturated DPPC/DPPG monolayers and otherwise led to lipid condensation. For Ag-NH, adsorption was driven primarily by electrostatic interactions with PGs. Analysis of the subphase Ag and phosphorus concentrations confirmed that Ag-NH had a higher degree binding compared to Ag-COOH, and that the monolayer response was not due to lipid extraction.

The acute respiratory distress syndrome (ARDS) is a highly lethal condition that impairs lung function and causes respiratory failure. Mechanical ventilation maintains gas exchange in patients with ARDS, but exposes lung cells to physical forces that exacerbate injury. Our data demonstrate that mTOR complex 1 (mTORC1) is a mechanosensor in lung epithelial cells and that activation of this pathway during mechanical ventilation impairs lung function. We found that mTORC1 is activated in lung epithelial cells following volutrauma and atelectrauma in mice and humanized in vitro models of the lung microenvironment. mTORC1 is also activated in lung tissue of mechanically ventilated patients with ARDS. Deletion of Tsc2, a negative regulator of mTORC1, in epithelial cells impairs lung compliance during mechanical ventilation.Conversely, treatment with rapamycin at the time mechanical ventilation is initiated improves lung compliance without altering lung inflammation or barrier permeability. mTORC1 inhibition mitigates physiologic lung injury by preventing surfactant dysfunction during mechanical ventilation. Our data demonstrate that in contrast to canonical mTORC1 activation under favorable growth conditions, activation of mTORC1 during mechanical ventilation exacerbates lung injury and inhibition of this pathway may be a novel therapeutic target to mitigate ventilator-induced lung injury during ARDS.

Background: Use of lungs donated after circulatory death (DCD) has expanded, but changes in donor/recipient characteristics and comparison to brain dead donors (DBD) has not been studied. We examined the evolution of the use of DCD lungs for transplantation and compare outcomes to DBD lungs.Methods: The SRTR database was used to construct three 5-year intervals. Perioperative variables and survival were compared by era and for DCD vs. DBD. Geographic variation was estimated using recipient permanent address.Results: 728 DCD and 27,205 DBD lung transplants were identified. DCD volume increased from Era 1 (n = 73) to Era 3 (n = 528), representing 1.1% and 4.2% of lung transplants. Proportionally more DCD recipients were in ICU or on ECMO pre-transplant, and had shorter waitlist times. DCD donors were older, had lower PaO2/FiO2 ratios compared to DBD, more likely to be bilateral, had longer ischemic time, length of stay, post-op dialysis, and increased use of lung perfusion. There was no difference in overall survival. Geographically, use was heterogeneous.Conclusion: DCD utilization is low but increasing. Despite increasing ischemic time and transplantation into sicker patients, survival is similar, which supports further DCD use in lung transplantation. DCD lung transplantation presents an opportunity to continue to expand the donor pool.