Dexamethasone and Trans-Dehydroandrosterone Significantly Reduce Pulmonary Epithelial Cell Injuries Associated with Mechanical Ventilation

Many patients who suffer from pulmonary diseases cannot inflate their lungs normally as they need Mechanical Ventilation (MV) to assist them. The stress associated with MV can damage delicate epithelium in small airways and alveoli which can cause complications in many cases resulting in Ventilation Induced Lungs injuries (VILI) especially in patients with Acute Respiratory Distress Syndrome (ARDS). Therefore, efforts have been used to developed safe modes for its use. In our work, we propose a different approach to decrease injuries of Epithelial Cells (EpCs) upon MV. We alter EpCs' cytoskeletal structure to increase their survival rate during airway reopening conditions associated with MV. We tested two anti-inflammatory drugs Dexamethasone (DEX) and Trans-Dehydroandrosterone (DHEA) to alter the cytoskeleton. Cultured rat L2 alveolar EpCs were exposed to airway reopening conditions using a parallel plate perfusion chamber. Cells were exposed to a single bubble propagation to simulate stresses associated with mechanical ventilation in both control and study groups. Cellular injury and cytoskeleton reorganization were assessed via fluorescent microscopy while cell topography was studied via Atomic Force Microscopy (AFM). Our results indicate that cells in cultured media, DEX, or DHEA solutions did not lead to cell death (static cultures). Bubble flows caused significant cell injury. Pre-exposure with DEX or DHEA decreased cell death significantly. The AFM verified cells' mechanics' alteration due to actin fibers depolymerization. These results suggest potential beneficial effects of DEX and DHEA for ARRDS treatment for COVID-19 patients. They are also critical for VILI and applicable to future clinical studies.

Nitric Oxide Releasing Hydrogel Nanoparticles Decreases Epithelial Cell Injuries Associated With Airway Reopening

Acute respiratory distress syndrome (ARDS) is an acute inflammatory lung condition. It is characterized by disruption of gas exchange inside the alveoli, accumulation of protein edema, and an increase in lung stiffness. One major cause of ARDS is a lung infection, such as SARS-COV-2 infection. Lungs of ARDS patients need to be mechanically ventilated for airway reopening. Consequently, ventilation might damage delicate lung tissue leading to excess edema, known as ventilator-induced lung injury (VILI). Mortality of COVID-19 patients under VILI seems to be higher than non-COVID patients, necessitating effective preventative therapies. VILI occurs when small air bubbles form in the alveoli, injuring epithelial cells (EPC) due to shear stress. Nitric oxide (NO) inhalation was suggested as a therapy for ARDS, however, it was shown that it is not effective because of the extremely short half-life of NO. In this study, NO-releasing nanoparticles were produced and tested in an in vitro model, representing airways in the deep lung. Cellular injuries were quantified via fluorescent live/dead assay. Atomic force microscopy (AFM) was used to assess cell morphology. qRT-PCR was performed to assess the expression of inflammatory markers, specifically IL6 and CCL2. ELISA was performed to assess IL6 and confirm qRT-PCR results at the protein level. Finally, ROS levels were assessed in all groups. Here, we show that NO delivery via nanoparticles enhanced EPC survival and recovery, AFM measurements revealed that NO exposure affect cell morphology, while qRT-PCR demonstrated a significant downregulation in IL6 and CCL2 expression when treating the cells to NO both before and after shear exposure. ELISA results for IL6 confirmed qRT-PCR data. ROS experiment results support our findings from previous experiments. These findings demonstrate that NO-releasing nanoparticles can be used as an effective delivery approach of NO to deep lung to prevent/reduce ARDS associated inflammation and cell injuries. This information is particularly useful to treat severe ARDS due to COVID-19 infection. These nanoparticles will be useful when clinically administrated to COVID-19 patients to reduce the symptoms originating from lung distress.

Influence of airway wall compliance on epithelial cell injury and adhesion during interfacial flows

Interfacial flows during cyclic airway reopening are an important source of ventilator-induced lung injury. However, it is not known how changes in airway wall compliance influence cell injury during airway reopening. We used an in vitro model of airway reopening in a compliant microchannel to investigate how airway wall stiffness influences epithelial cell injury. Epithelial cells were grown on gel substrates with different rigidities, and cellular responses to substrate stiffness were evaluated in terms of metabolic activity, mechanics, morphology, and adhesion. Repeated microbubble propagations were used to simulate cyclic airway reopening, and cell injury and detachment were quantified via live/dead staining. Although cells cultured on softer gels exhibited a reduced elastic modulus, these cells experienced less plasma membrane rupture/necrosis. Cells on rigid gels exhibited a minor, but statistically significant, increase in the power law exponent and also exhibited a significantly larger height-to-length aspect ratio. Previous studies indicate that this change in morphology amplifies interfacial stresses and, therefore, correlates with the increased necrosis observed during airway reopening. Although cells cultured on stiff substrates exhibited more plasma membrane rupture, these cells experienced significantly less detachment and monolayer disruption during airway reopening. Western blotting and immunofluorescence indicate that this protection from detachment and monolayer disruption correlates with increased focal adhesion kinase and phosphorylated paxillin expression. Therefore, changes in cell morphology and focal adhesion structure may govern injury responses during compliant airway reopening. In addition, these results indicate that changes in airway compliance, as occurs during fibrosis or emphysema, may significantly influence cell injury during mechanical ventilation.

The unusual symmetric reopening effect induced by pulmonary surfactant

This study investigates the stability of a finger of air as it propagates into a liquid-filled model of a liquid-filled model of a pulmonary bifurcation. We seek to elucidate the stability characteristics of the reopening of daughter airways, an event that may be important to the treatment of acute lung disease. To do so, we investigated the symmetry of reopening under conditions of nearly constant surface tension with 1) purified H2O or 2) an anionic surfactant (sodium dodecyl sulfate). Dynamic surface tension was investigated using pulmonary surfactant (Infasurf) with and without the presence of albumin. Flow visualization was accomplished using a microparticle image velocimetry (μ-PIV)/shadowgraph system through which we measured 1) the propagation velocity of the finger of air that reopens each daughter branch, and 2) the instantaneous and averaged velocity field of liquid phase surrounding the tip of the propagating bubble. Only pulmonary surfactant demonstrated the ability of maintaining a nearly symmetric propagation in the daughter channels, which is likely to lead to homogeneous airway reopening. In contrast, when pulmonary surfactant was inactivated by albumin or when the system was held at a nearly constant surface tension, reopening occurred asymmetrically. Our analysis suggests that Infasurf's dynamic surface tension qualities are important to stabilize the removal of liquid obstructions. This demonstrates a new important function of pulmonary surfactant for airway reopening of a multibranched network.

The influence of surfactant on the propagation of a semi-infinite bubble through a liquid-filled compliant channel

We investigate the influence of a soluble surfactant on the steady-state motion of a finger of air through a compliant channel. This study provides a basic model from which to understand the fluid–structure interactions and physicochemical hydrodynamics of pulmonary airway reopening. Airway closure occurs in lung diseases such as respiratory distress syndrome and acute respiratory distress syndrome as a result of fluid accumulation and surfactant insufficiency. This results in ‘compliant collapse’ with the airway walls buckled and held in apposition by a liquid occlusion that blocks the passage of air. Airway reopening is essential to the recovery of adequate ventilation, but has been associated with ventilator-induced lung injury because of the exposure of airway epithelial cells to large interfacial flow-induced pressure gradients. Surfactant replacement is helpful in modulating this deleterious mechanical stimulus, but is limited in its effectiveness owing to slow surfactant adsorption. We investigate the effect of surfactant on micro-scale models of reopening by computationally modelling the steady two-dimensional motion of a semi-infinite bubble propagating through a liquid-filled compliant channel doped with soluble surfactant. Many dimensionless parameters affect reopening, but we primarily investigate how the reopening pressure ${p}_{b} $ depends upon the capillary number $\mathit{Ca}$ (the ratio of viscous to surface tension forces), the adsorption depth parameter $\lambda $ (a bulk concentration parameter) and the bulk Péclet number ${\mathit{Pe}}_{b} $ (the ratio of bulk convection to diffusion). These studies demonstrate a dependence of ${p}_{b} $ on $\lambda $, and suggest that a critical bulk concentration must be exceeded to operate as a low-surface-tension system. Normal and tangential stress gradients remain largely unaffected by physicochemical interactions – for this reason, further biological studies are suggested that will clarify the role of wall flexibility and surfactant on the protection of the lung from atelectrauma.