Clathrin-Mediated Albumin Clearance in Alveolar Epithelial Cells of Murine Precision-Cut Lung Slices.

A hallmark of acute respiratory distress syndrome (ARDS) is an accumulation of protein-rich alveolar edema that impairs gas exchange and leads to worse outcomes. Thus, understanding the mechanisms of alveolar albumin clearance is of high clinical relevance. Here, we investigated the mechanisms of the cellular albumin uptake in a three-dimensional culture of precision-cut lung slices (PCLS). We found that up to 60% of PCLS cells incorporated labeled albumin in a time- and concentration-dependent manner, whereas virtually no uptake of labeled dextran was observed. Of note, at a low temperature (4 °C), saturating albumin receptors with unlabeled albumin and an inhibition of clathrin-mediated endocytosis markedly decreased the endocytic uptake of the labeled protein, implicating a receptor-driven internalization process. Importantly, uptake rates of albumin were comparable in alveolar epithelial type I (ATI) and type II (ATII) cells, as assessed in PCLS from a SftpcCreERT2/+: tdTomatoflox/flox mouse strain (defined as EpCAM+CD31-CD45-tdTomatoSPC-T1α+ for ATI and EpCAM+CD31-CD45-tdTomatoSPC+T1α- for ATII cells). Once internalized, albumin was found in the early and recycling endosomes of the alveolar epithelium as well as in endothelial, mesenchymal, and hematopoietic cell populations, which might indicate transcytosis of the protein. In summary, we characterize albumin uptake in alveolar epithelial cells in the complex setting of PCLS. These findings may open new possibilities for pulmonary drug delivery that may improve the outcomes for patients with respiratory failure.

Development of human-derived, three-dimensional respiratory epithelial tissue constructs with perfusable microvasculature on a high-throughput microfluidics screening platform.

The COVID-19 pandemic has highlighted the need for human respiratory tract-based assay platforms for efficient discovery and development of antivirals and disease-modulating therapeutics. Physiologically relevant tissue models of the lower respiratory tract (LRT), including the respiratory bronchioles and the alveolar sacs, are of high interest because they are the primary site of severe SARS-CoV-2 infection and are most affected during the terminal stage of COVID-19. Current epithelial lung models used to study respiratory viral infections include lung epithelial cells at the air-liquid interface (ALI) with fibroblasts and endothelial cells, but such models do not have a perfusable microvascular network to investigate both viral infectivity and viral infection-induced thrombotic events. Using a high throughput, 64-chip microfluidic plate-based platform, we have developed two novel vascularized, LRT multi-chip models for the alveoli and the small airway. Both models include a perfusable microvascular network consisting of human primary microvascular endothelial cells, fibroblasts and pericytes. The established biofabrication protocols also enable the formation of differentiated lung epithelial layers at the ALI on top of the vascularized tissue bed. We validated the physiologically relevant cellular composition, architecture and perfusion of the vascularized lung tissue models using fluorescence microscopy, flow cytometry, and electrical resistance measurements. These vascularized, perfusable microfluidic lung tissue on high throughput assay platforms will enable the development of respiratory viral infection and disease models for research investigation and drug discovery.

Systematic Review of Hematological Finding in Critical Covid-19 patients

Severe COVID-19 infection results in bilateral interstitial pneumonia, often leading to acute respiratory distress syndrome (ARDS) and pulmonary fibrosis in survivors. Most patients with severe COVID-19 infections who died had developed ARDS. Currently, ARDS is treated with supportive measures, but regenerative medicine approaches including extracellular vesicle (EV)-based therapies have shown promise. Herein, we aimed to analyze whether EV-based therapies could be effective in treating severe pulmonary conditions that affect COVID-19 patients and to understand their relevance for an eventual therapeutic application to human patients. Using a defined search strategy, we conducted a systematic review of the literature and found 39 articles (2014-2020) that reported effects of EVs, mainly derived from stem cells, in lung injury models (one large animal study, none in human). EV treatment resulted in: (1) Attenuation of inflammation (reduction of pro-inflammatory cytokines and neutrophil infiltration, M2 macrophage polarization);(2) Regeneration of alveolar epithelium (decreased apoptosis and stimulation of surfactant production);(3) Repair of microvascular permeability (increased endothelial cell junction proteins);(4) Prevention of fibrosis (reduced fibrin production). These effects were mediated by the release of EV cargo and identified factors including mi Rs-126, 30b-3p, 145, 27a-3p, syndecan-1, hepatocyte growth factor and angiopoietin-1. This review indicates that EV-based therapies hold great potential for COVID-19 related lung injuries as they target multiple pathways and enhance tissue regeneration. However, before translating EV therapies into human clinical trials, efforts should be directed at developing good manufacturing practice solutions for EVs and testing optimal dosage and administration route in large animal models.

Molecular mechanisms of Na,K-ATPase dysregulation driving alveolar epithelial barrier failure in severe COVID-19.

A significant number of patients with coronavirus disease 2019 (COVID-19) develop acute respiratory distress syndrome (ARDS) that is associated with a poor outcome. The molecular mechanisms driving failure of the alveolar barrier upon severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection remain incompletely understood. The Na,K-ATPase is an adhesion molecule and a plasma membrane transporter that is critically required for proper alveolar epithelial function by both promoting barrier integrity and resolution of excess alveolar fluid, thus enabling appropriate gas exchange. However, numerous SARS-CoV-2-mediated and COVID-19-related signals directly or indirectly impair the function of the Na,K-ATPase, thereby potentially contributing to disease progression. In this Perspective, we highlight some of the putative mechanisms of SARS-CoV-2-driven dysfunction of the Na,K-ATPase, focusing on expression, maturation, and trafficking of the transporter. A therapeutic mean to selectively inhibit the maladaptive signals that impair the Na,K-ATPase upon SARS-CoV-2 infection might be effective in reestablishing the alveolar epithelial barrier and promoting alveolar fluid clearance and thus advantageous in patients with COVID-19-associated ARDS.

Modulating the Barrier Function of Human Alveolar Epithelial (hAELVi) Cell Monolayers as a Model of Inflammation.

The incidence of inflammatory lung diseases such as acute respiratory distress syndrome (ARDS) remains an important problem, particularly in the present time with the Covid-19 pandemic. However, an adequate in vitro test system to monitor the barrier function of the alveolar epithelium during inflammation and for assessing anti-inflammatory drugs is urgently needed. Therefore, we treated human Alveolar Epithelial Lentivirus-immortalised cells (hAELVi cells) with the pro-inflammatory cytokines TNF-α (25 ng/ml) and IFN-γ (30 ng/ml), in the presence or absence of hydrocortisone (HC). While TNF-α and IFN-γ are known to reduce epithelial barrier properties, HC could be expected to protect the barrier function and result in an anti-inflammatory effect. We investigated the impact of anti-inflammatory/inflammatory treatment on transepithelial electrical resistance (TEER) and the apparent permeability coefficient (Papp) of the low permeability marker sodium fluorescein (NaFlu). After incubating hAELVi cells for 48 hours with a combination of TNF-α and IFN-γ, there was a significant decrease in TEER and a significant increase in the Papp. The presence of HC maintained the TEER values and barrier properties, so that no significant Papp change was observed. By using hAELVi cells to study anti-inflammatory drugs in vitro, the need for animal experiments could be reduced and pulmonary drug development accelerated.

Activation of the SARS-CoV-2 Receptor Ace2 through JAK/STAT-Dependent Enhancers during Pregnancy.

ACE2 binds the coronavirus SARS-CoV-2 and facilitates its cellular entry. Interferons activate ACE2 expression in pneumocytes, suggesting a critical role of cytokines in SARS-CoV-2 target cells. Viral RNA was detected in breast milk in at least seven studies, raising the possibility that ACE2 is expressed in mammary tissue during lactation. Here, we show that Ace2 expression in mouse mammary tissue is induced during pregnancy and lactation, which coincides with the activation of intronic enhancers. These enhancers are occupied by the prolactin-activated transcription factor STAT5 and additional regulatory factors, including RNA polymerase II. Deletion of Stat5a results in decommissioning of the enhancers and an 83% reduction of Ace2 mRNA. We also demonstrate that Ace2 expression increases during lactation in lung, but not in kidney and intestine. JAK/STAT components are present in a range of SARS-CoV-2 target cells, opening the possibility that cytokines contribute to the viral load and extrapulmonary pathophysiology.