Remodeling of Murine Branch Pulmonary Arteries Under Chronic Hypoxia and Short-Term Normoxic Recovery.

Chronic hypoxia plays a central role in diverse pulmonary pathologies, but its effects on longitudinal changes in the biomechanical behavior of proximal pulmonary arteries remain poorly understood. Similarly, effects of normoxic recovery have not been well studied. Here, we report hypoxia-induced changes in composition, vasoactivity, and passive biaxial mechanics in the main branch pulmonary artery of male C57BL/6J mice exposed to 10% FiO2 for 1, 2, or 3 weeks. We observed significant changes in extracellular matrix, and consequently wall mechanics, as early as 1 week of hypoxia. While circumferential stress and stiffness returned toward normal values by 2-3 weeks of hypoxia, area fractions of cytoplasm and thin collagen fibers did not return toward normal until after 1 week of normoxic recovery. By contrast, elastic energy storage and overall distensibility remained reduced after 3 weeks of hypoxia as well as following 1 week of normoxic recovery. While smooth muscle and endothelial cell responses were attenuated under hypoxia, smooth muscle but not endothelial cell responses recovered following 1 week of subsequent normoxia. Collectively, these data suggest that homeostatic processes were unable to preserve or restore overall function, at least over a brief period of normoxic recovery. Longitudinal changes are critical in understanding large pulmonary artery remodeling under hypoxia, and its reversal, and will inform predictive models of vascular adaptation.

Developmental changes in lung function of mice are independent of sex as a biological variable.

Pulmonary function testing (PFT) in mice includes biomechanical assessment of lung function relevant to physiology in health and its alteration in disease, hence, it is frequently used in preclinical modeling of human lung pathologies. Despite numerous reports of PFT in mice of various ages, there is a lack of reference data for developing mice collected using consistent methods. Therefore, we profiled PFTs in male and female C57BL/6J mice from 2 to 23 wk of age, providing reference values for age- and sex-dependent changes in mouse lung biomechanics during development and young adulthood. Although males and females have similar weights at birth, females weigh significantly less than males after 5 wk of age (P < 0.001) with largest weight gain observed between 3 and 8 wk in females and 3 and 13 wk in males, after which weight continued to increase more slowly up to 23 wk of age. Lung function parameters including static compliance and inspiratory capacity also increased rapidly between 3 and 8 wk in female and male mice, with male mice having significantly greater static compliance and inspiratory capacity than female mice (P < 0.001). Although these parameters appear higher in males at a given age, allometric scaling showed that static compliance and inspiratory compliance were comparable between the two sexes. This suggests that differences in measurements of lung function are likely body weight-based rather than sex-based. We expect these data to facilitate future lung disease research by filling a critical knowledge gap in our field.NEW & NOTEWORTHY This study provides reference values for changes in mouse lung biomechanics from 2 to 23 wk of age. There are rapid developmental changes in lung structure and function of male and female mice between the ages of 3 and 8 wk. Male mice become noticeably heavier than female mice at or about 5 wk of age. We identified that differences in normal lung function measurements are likely weight-based, not sex-based.

Prognostic variables and decannulation of tracheostomy in the long-term acute care environment: a case for clinician-driven decision-making.

Purpose: Tracheostomy is a necessary procedure required for prolonged mechanical ventilation in long-term acute care hospitals (LTACH). Many factors influence successful decannulation, or tracheostomy removal, and it is unclear what factors are essential for determining decannulation. The purpose of this study was to determine retrospective performance of single prognostic variables for successful decannulation, like peak expiratory flow measurement, overnight oximetry testing, and blood gas analysis. Methods: A retrospective analysis of a three-year period to investigate the association between peak flow (PF) measurements ≥160 L/min, successful overnight oximetry (ONO), sex, and decannulation success. Average PF measurements, arterial blood gas (ABG), days on mechanical ventilation, LTACH length of stay (LOS), and age were also investigated. Results: We examined the records of 135 patients, 127 of which were successfully decannulated. PF measurements ≥160 L/min (p=0.16), sex (p<0.05) and passing ONO (p<0.05) were significantly different between successfully and unsuccessfully decannulated patients; mean ABG (pH, pCO2, pO2), mechanical ventilation days, LOS, and age were not significantly different (p>0.05). Conclusions: These results suggest no single prognostic variable can predict decannulation outcomes. Rather, clinical judgment of experienced medical professionals appears sufficient to achieve a 94% decannulation success rate. Additional investigation is required to determine what metrics are necessary, or if clinical judgment alone can predict decannulation success.

A Multi-omic Analysis of the Human Lung Reveals Distinct Cell Specific Aging and Senescence Molecular Programs.

Age is a major risk factor for lung disease. To understand the mechanisms underlying this association, we characterized the changing cellular, genomic, transcriptional, and epigenetic landscape of lung aging using bulk and single-cell RNAseq (scRNAseq) data. Our analysis revealed age-associated gene networks that reflected hallmarks of aging, including mitochondrial dysfunction, inflammation, and cellular senescence. Cell type deconvolution revealed age-associated changes in the cellular composition of the lung: decreased alveolar epithelial cells and increased fibroblasts and endothelial cells. In the alveolar microenvironment, aging is characterized by decreased AT2B cells and reduced surfactant production, a finding that was validated by scRNAseq and IHC. We showed that a previously reported senescence signature, SenMayo, captures cells expressing canonical senescence markers. SenMayo signature also identified cell-type specific senescence-associated co-expression modules that have distinct molecular functions, including ECM regulation, cell signaling, and damage response pathways. Analysis of somatic mutations showed that burden was highest in lymphocytes and endothelial cells and was associated with high expression of senescence signature. Finally, aging and senescence gene expression modules were associated with differentially methylated regions, with inflammatory markers such as IL1B, IL6R, and TNF being significantly regulated with age. Our findings provide new insights into the mechanisms underlying lung aging and may have implications for the development of interventions to prevent or treat age-related lung diseases.

The association between beta-natriuretic peptide (BNP) serum levels and liberation from mechanical ventilation.

Prolonged time on mechanical ventilation is associated with multiple consequences for both the patient and medical facility. Based on anecdotal evidence that sustained elevation of beta-natriuretic protein (BNP) during a patient's stay in a long-term acute care hospital (LTACH) was associated with failure to wean from prolonged mechanical ventilation, we investigated if there is an association between a decrease in BNP levels during one's stay and successful weaning from prolonged mechanical ventilation. We performed a retrospective study of 66 patient records revealing no correlation between lowering BNP levels and probability of liberating a patient from prolonged mechanical ventilation in an LTACH environment where the probability of liberation from mechanical ventilation is high (> 85%). BNP measurements by itself does not appear to be a helpful tool in the likelihood of liberation from mechanical ventilation AUC = 0.61 (CI: 0.48-0.72). In an LTACH setting with high success rates of liberation from mechanical ventilation, it does not appear to be necessary to trend BNP measurements in attempts to liberate patients from prolonged mechanical ventilation.

Epidermal Growth Factor Receptor Inhibition Is Protective in Hyperoxia-Induced Lung Injury.

Aims: Studies have linked severe hyperoxia, or prolonged exposure to very high oxygen levels, with worse clinical outcomes. This study investigated the role of epidermal growth factor receptor (EGFR) in hyperoxia-induced lung injury at very high oxygen levels (>95%). Results: Effects of severe hyperoxia (100% oxygen) were studied in mice with genetically inhibited EGFR and wild-type littermates. Despite the established role of EGFR in lung repair, EGFR inhibition led to improved survival and reduced acute lung injury, which prompted an investigation into this protective mechanism. Endothelial EGFR genetic knockout did not confer protection. EGFR inhibition led to decreased levels of cleaved caspase-3 and poly (ADP-ribosyl) polymerase (PARP) and decreased terminal dUTP nick end labeling- (TUNEL-) positive staining in alveolar epithelial cells and reduced ERK activation, which suggested reduced apoptosis in vivo. EGFR inhibition decreased hyperoxia (95%)-induced apoptosis and ERK in murine alveolar epithelial cells in vitro, and CRISPR-mediated EGFR deletion reduced hyperoxia-induced apoptosis and ERK in human alveolar epithelial cells in vitro. Innovation. This work defines a protective role of EGFR inhibition to decrease apoptosis in lung injury induced by 100% oxygen. This further characterizes the complex role of EGFR in acute lung injury and outlines a novel hyperoxia-induced cell death pathway that warrants further study. Conclusion: In conditions of severe hyperoxia (>95% for >24 h), EGFR inhibition led to improved survival, decreased lung injury, and reduced cell death. These findings further elucidate the complex role of EGFR in acute lung injury.

Compromised Cardiopulmonary Function in Fibulin-5 Deficient Mice.

Competent elastic fibers are critical to the function of the lung and right circulation. Murine models of elastopathies can aid in understanding the functional roles of the elastin and elastin-associated glycoproteins that constitute elastic fibers. Here, we quantify together lung and pulmonary arterial structure, function, and mechanics with right heart function in a mouse model deficient in the elastin-associated glycoprotein fibulin-5. Differences emerged as a function of genotype, sex, and arterial region. Specifically, functional studies revealed increased lung compliance in fibulin-5 deficiency consistent with a histologically observed increased alveolar disruption. Biaxial mechanical tests revealed that the primary branch pulmonary arteries exhibit decreased elastic energy storage capacity and wall stress despite only modest differences in circumferential and axial material stiffness in the fibulin-5 deficient mice. Histological quantifications confirm a lower elastic fiber content in the fibulin-5 deficient pulmonary arteries, with fragmented elastic laminae in the outer part of the wall - likely the reason for reduced energy storage. Ultrasound measurements confirm sex differences in compromised right ventricular function in the fibulin-5 deficient mice. These results reveal compromised right heart function, but opposite effects of elastic fiber dysfunction on the lung parenchyma (significantly increased compliance) and pulmonary arteries (trend toward decreased distensibility), and call for further probing of ventilation-perfusion relationships in pulmonary pathologies. Amongst many other models, fibulin-5 deficient mice can contribute to our understanding of the complex roles of elastin in pulmonary health and disease.

Mechanisms of Hypoxia-Induced Pulmonary Arterial Stiffening in Mice Revealed by a Functional Genetics Assay of Structural, Functional, and Transcriptomic Data.

Hypoxia adversely affects the pulmonary circulation of mammals, including vasoconstriction leading to elevated pulmonary arterial pressures. The clinical importance of changes in the structure and function of the large, elastic pulmonary arteries is gaining increased attention, particularly regarding impact in multiple chronic cardiopulmonary conditions. We establish a multi-disciplinary workflow to understand better transcriptional, microstructural, and functional changes of the pulmonary artery in response to sustained hypoxia and how these changes inter-relate. We exposed adult male C57BL/6J mice to normoxic or hypoxic (FiO2 10%) conditions. Excised pulmonary arteries were profiled transcriptionally using single cell RNA sequencing, imaged with multiphoton microscopy to determine microstructural features under in vivo relevant multiaxial loading, and phenotyped biomechanically to quantify associated changes in material stiffness and vasoactive capacity. Pulmonary arteries of hypoxic mice exhibited an increased material stiffness that was likely due to collagen remodeling rather than excessive deposition (fibrosis), a change in smooth muscle cell phenotype reflected by decreased contractility and altered orientation aligning these cells in the same direction as the remodeled collagen fibers, endothelial proliferation likely representing endothelial-to-mesenchymal transitioning, and a network of cell-type specific transcriptomic changes that drove these changes. These many changes resulted in a system-level increase in pulmonary arterial pulse wave velocity, which may drive a positive feedback loop exacerbating all changes. These findings demonstrate the power of a multi-scale genetic-functional assay. They also highlight the need for systems-level analyses to determine which of the many changes are clinically significant and may be potential therapeutic targets.

Integrated Single-Cell Atlas of Endothelial Cells of the Human Lung.

BACKGROUND: Cellular diversity of the lung endothelium has not been systematically characterized in humans. We provide a reference atlas of human lung endothelial cells (ECs) to facilitate a better understanding of the phenotypic diversity and composition of cells comprising the lung endothelium. METHODS: We reprocessed human control single-cell RNA sequencing (scRNAseq) data from 6 datasets. EC populations were characterized through iterative clustering with subsequent differential expression analysis. Marker genes were validated by fluorescent microscopy and in situ hybridization. scRNAseq of primary lung ECs cultured in vitro was performed. The signaling network between different lung cell types was studied. For cross-species analysis or disease relevance, we applied the same methods to scRNAseq data obtained from mouse lungs or from human lungs with pulmonary hypertension. RESULTS: Six lung scRNAseq datasets were reanalyzed and annotated to identify >15 000 vascular EC cells from 73 individuals. Differential expression analysis of EC revealed signatures corresponding to endothelial lineage, including panendothelial, panvascular, and subpopulation-specific marker gene sets. Beyond the broad cellular categories of lymphatic, capillary, arterial, and venous ECs, we found previously indistinguishable subpopulations; among venous EC, we identified 2 previously indistinguishable populations: pulmonary-venous ECs (COL15A1neg) localized to the lung parenchyma and systemic-venous ECs (COL15A1pos) localized to the airways and the visceral pleura; among capillary ECs, we confirmed their subclassification into recently discovered aerocytes characterized by EDNRB, SOSTDC1, and TBX2 and general capillary EC. We confirmed that all 6 endothelial cell types, including the systemic-venous ECs and aerocytes, are present in mice and identified endothelial marker genes conserved in humans and mice. Ligand-receptor connectome analysis revealed important homeostatic crosstalk of EC with other lung resident cell types. scRNAseq of commercially available primary lung ECs demonstrated a loss of their native lung phenotype in culture. scRNAseq revealed that endothelial diversity is maintained in pulmonary hypertension. Our article is accompanied by an online data mining tool (www.LungEndothelialCellAtlas.com). CONCLUSIONS: Our integrated analysis provides a comprehensive and well-crafted reference atlas of ECs in the normal lung and confirms and describes in detail previously unrecognized endothelial populations across a large number of humans and mice.

Disinfection of Pseudomonas aeruginosa from N95 respirators with ozone: a pilot study.

INTRODUCTION: Personal protective equipment shortages require the reuse of N95 respirators. We sought the necessary conditions for ozone to disinfect N95 respirators for reuse and the effects of multiple cycles of exposure. METHODS: Portions of 3M 1870 N95 respirators were exposed to ozone at 400 ppm with 80% humidity for 2 hours to determine effectiveness of ozone on killing Pseudomonas aeruginosa. Entire 3M 1870 N95 respirators were exposed to five cycles of 400 ppm with 80% or higher humidity for 2 hours then evaluated for ozone's effects on airflow resistance, filtration efficiency, strap strength and quantitative fit. RESULTS: Ozone exposure disinfected 3M 1870 N95 respirators heavily inoculated with P. aeruginosa. Ozone exposure did not negatively affect the airflow resistance, filtration efficiency, strap strength or fit of the 3M 1870 N95 respirator. DISCUSSION: These results suggest that ozone is a feasible strategy to disinfect N95 respirators for reuse during this and future pandemics.

Plasma mitochondrial DNA is associated with extrapulmonary sarcoidosis.

Sarcoidosis is an unpredictable granulomatous disease in which African Americans disproportionately experience aggressive phenotypes. Mitochondrial DNA (mtDNA) released by cells in response to various stressors contributes to tissue remodelling and inflammation. While extracellular mtDNA has emerged as a biomarker in multiple diseases, its relevance to sarcoidosis remains unknown. We aimed to define an association between extracellular mtDNA and clinical features of sarcoidosis.Extracellular mtDNA concentrations were measured using quantitative PCR for the human MT-ATP6 gene in bronchoalveolar (BAL) and plasma samples from healthy controls and patients with sarcoidosis from The Yale Lung Repository; associations between MT-ATP6 concentrations and Scadding stage, extrapulmonary disease and demographics were sought. Results were validated in the Genomic Research in Alpha-1 Antitrypsin Deficiency and Sarcoidosis cohort.Relative to controls, MT-ATP6 concentrations in sarcoidosis subjects were robustly elevated in the BAL fluid and plasma, particularly in the plasma of patients with extrapulmonary disease. Relative to Caucasians, African Americans displayed excessive MT-ATP6 concentrations in the BAL fluid and plasma, for which the latter compartment correlated with significantly higher odds of extrapulmonary disease.Enrichments in extracellular mtDNA in sarcoidosis are associated with extrapulmonary disease and African American descent. Further study into the mechanistic basis of these clinical findings may lead to novel pathophysiologic and therapeutic insights.

Pulmonary artery pseudoaneurysm causing massive hemoptysis in hyperimmunoglobulin E syndrome: a case report.

BACKGROUND: Hyperimmunoglobulin E syndrome (HIES) is a rare primary immunodeficiency disorder defined by high serum immunoglobulin E titers that is associated with recurrent respiratory infections, formation of pneumoatoceles, recurrent skin abscesses, and characteristic dental and skeletal abnormalities. CASE PRESENTATION: We report a case of a 56-year-old male with a history of HIES, cavitary mycetomas, and allergic bronchopulmonary aspergillosis who presented with recurrent massive hemoptysis. Bronchial artery angiography and bronchoscopy failed to identify active hemorrhage, and two embolizations of the bronchial artery did not resolve the bleeding. Subsequently, selective pulmonary artery angiography was conducted that demonstrated a subsegmental pulmonary artery branch pseudoaneurysm with extravasation into an adjacent lung cavity. This was treated successfully with transcatheter embolization. CONCLUSIONS: To our knowledge, this is the first case reported of pulmonary artery pseudoaneurysm in HIES in the medical literature. Pulmonary artery pseudoaneurysm should be considered in the differential diagnosis in patients with HIES and massive hemoptysis.

Early and prolonged opportunities to practice suturing increases medical student comfort with suturing during clerkships: Suturing during cadaver dissection.

Medical students are expected to perform common procedures such as suturing on patients during their third-year clerkships. However, these experiences are often viewed by medical students as stressors rather than opportunities for learning. The source of this stress is the lack of instruction on common procedures prior to being asked to observe or perform the procedure on a patient. First-time exposures to procedures in stressful environments may result in decreased confidence in medical students and decrease the frequency with which they perform these procedures in the future. The authors sought to change this paradigm by: (1) introducing a suturing module to first-year medical students in the context of the anatomy dissection laboratory and (2) measuring its effects on student attitudes and behavior over the course of their third-year clerkships when they encounter patients. The authors found that early and prolonged introduction to suturing was associated with increased student confidence relative to suturing a patient. Participation in the suturing module was associated with increased student confidence in identifying suturing instruments (P < 0.001) and suturing patients (P = 0.013). Further it positively affected their behavior as demonstrated by increased performance of suturing events from students exposed to the suturing module. (P < 0.001) This study demonstrates that early and prolonged opportunities to practice a procedural skill in a low-stress environment increases student confidence during patient interactions and alters student behavior.

Central Nervous System Oxygen Toxicity and Hyperbaric Oxygen Seizures.

INTRODUCTION: The use of hyperbaric oxygen (O2) as a therapeutic agent carries with it the risk of central nervous system (CNS) O2 toxicity. METHODS: To further the understanding of this risk and the nature of its molecular mechanism, a review was conducted on the literature from various fields. RESULTS: Numerous physiological changes are produced by increased partial pressures of oxygen (Po2), which may ultimately result in CNS O2 toxicity. The human body has several equilibrated safeguards that minimize effects of reactive species on neural networks, believed to play a primary role in CNS O2 toxicity. Increased partial pressure of oxygen (Po2) appears to saturate protective enzymes and unfavorably shift protective reactions in the direction of neural network overstimulation. Certain regions of the CNS appear more susceptible than others to these effects. Failure to decrease the elevated Po2 can result in a tonic-clonic seizure and death. Randomized, controlled studies in human populations would require a multicenter trial over a long period of time with numerous endpoints used to identify O2 toxicity. CONCLUSIONS: The mounting scientific evidence and apparent increase in the number of hyperbaric O2 treatments demonstrate a need for further study in the near future.

PhytobezoarInduced Small Bowel Obstruction in a Young Male with Virgin Abdomen.

Phytobezoars are a rare cause of small bowel obstruction. Such cases are most commonly associated with previous abdominal surgery or poor dentition or psychiatric conditions. A 40 year old man with a virgin abdomen and excellent dentition and no underlying psychiatric condition presented with an acute abdomen. CT scan revealed a transition point between dilated proximal loops of small bowel and collapsed distal loops. Exploratory laparotomy revealed a phytobezoar unable to be milked into the cecum and an enterectomy with primary anastamosis was performed without complication. A detailed history revealing several less common predisposing factors for phytobezoars should increase clinical suspicion of a phytobezoarinduced small bowel obstruction in the setting of an acute abdomen. Vigilance in presentations of an acute abdomen improves the usefulness of medical imaging, such as a CT, to detect phytobezoars. Understanding mechanisms of phytobezoar formation helps guide management and may prevent surgery.