Hypercapnia alters stroma-derived Wnt production to limit ß-catenin signaling and proliferation in AT2 cells.

Persistent symptoms and radiographic abnormalities suggestive of failed lung repair are among the most common symptoms in patients with COVID-19 after hospital discharge. In mechanically ventilated patients with acute respiratory distress syndrome (ARDS) secondary to SARS-CoV-2 pneumonia, low tidal volumes to reduce ventilator-induced lung injury necessarily elevate blood CO2 levels, often leading to hypercapnia. The role of hypercapnia on lung repair after injury is not completely understood. Here - using a mouse model of hypercapnia exposure, cell lineage tracing, spatial transcriptomics, and 3D cultures - we show that hypercapnia limits ß-catenin signaling in alveolar type II (AT2) cells, leading to their reduced proliferative capacity. Hypercapnia alters expression of major Wnts in PDGFRα+ fibroblasts from those maintaining AT2 progenitor activity toward those that antagonize ß-catenin signaling, thereby limiting progenitor function. Constitutive activation of ß-catenin signaling in AT2 cells or treatment of organoid cultures with recombinant WNT3A protein bypasses the inhibitory effects of hypercapnia. Inhibition of AT2 proliferation in patients with hypercapnia may contribute to impaired lung repair after injury, preventing sealing of the epithelial barrier and increasing lung flooding, ventilator dependency, and mortality.

Lung Transplantation Promotes Survival In Patients with COVID-19 ARDS Who Require Extended Duration of VV ECMO support

Background: Veno-venous (VV) extracorporeal membrane oxygenation (ECMO) is increasingly being utilized to manage critical COVID-19 associated ARDS (CCAA) in patients who fail medical optimization and mechanical ventilatory support. The aim of this study was to determine the probability of weaning patients from ECMO over time and whether a subset of patients should be considered for lung transplantation. Additionally, we investigated when lung transplant should be considered after VV ECMO support. Methods: 49 patients with CCAA who required ECMO between January 2020 and September 2021 were investigated. Baseline patient demographics, clinical, laboratory, and follow-up data were compared. The change in probability of ECMO weaning based on duration of ECMO support was studied using a univariate analysis. Additionally, patients who received lung transplantation following VV ECMO for COVID-19 during this same period were studied to compare outcomes to those of patients with only VV ECMO support. Cox proportion hazard analysis was performed to determine predictors of survival in patients who required greater than 28 days of ECMO support. Yuden index was used to determine change in probability of survival with time on ECMO. Results: Of 49 patients, 17 (35%) received lung transplants and 32 (65%) remained on ECMO for >28 days. The probability of weaning patients from ECMO was highest within the first 10 days (60%);beyond 40 days, it was 5.1% (Fig. A). The probability of successfully weaning patients from ECMO significantly decreased over time and ECMO support greater than 28 days (Yuden index, Hazard ratio: 1.09, 95% CI;1.00-1.03) was associated with a significantly increased risk of mortality. Additionally, both survival to hospital discharge (p<0.001, Fig. B) and post-discharge survival (p<0.001, Fig. C) were significantly greater in those who were weaned from ECMO prior to 28 days than those who were weaned after 28 days. In those who could not be weaned from ECMO, lung transplantation (HR:0.47, p<0.01, 95% CI 0.17-0.94), ECMO duration (HR:1.09, p=0.01, 95% CI 1.00-1.03) and higher BUN levels (HR:1.02, p<0.01, 95% CI 1.01- 1.46) prior to ECMO initiation were independent predictors of survival. ECMO support of greater than 8 days was associated with a statistically significant increase in mortality compared to those who received fewer than 8 days of support (Yuden index, HR 1.96, CI 1.06-5.51). Furthermore, the projected survival of patients on ECMO support for greater than 8 days was substantially worse than those requiring fewer than 8 days of support (Fig. C and D). Conclusion: This study suggests that survival and accompanying lung recovery is more probable in patients who require a short duration of ECMO support whereas those who require longer durations, particularly exceeding 28 days, is associated with a lower rate of survival. (Figure Presented).

Hypercapnia Increases ACE2 Protein Expression and Pseudo-SARSCoV- 2 Virus Entry in Airway Epithelial Cells by Augmenting Cellular Cholesterol

Rationale: Individuals with COPD who develop COVID-19 are at increased risk of hospitalization, ICU admission and death. COPD is associated with increased airway epithelial expression of ACE2, the receptor mediating SARS-CoV-2 entry into cells. Hypercapnia commonly develops in advanced COPD and is associated with frequent and potentially fatal pulmonary infections. We previously reported that hypercapnia increases viral replication, lung injury and mortality in mice infected with influenza A virus. Also, global gene expression profiling of primary human bronchial epithelial (HBE) cells showed that elevated CO2 upregulates expression of cholesterol biosynthesis genes, including HMGCS1, and downregulates ATP-binding cassette (ABC) transporters that promote cholesterol efflux. Given that cellular cholesterol is important for entry of viruses into cells, in the current study we assessed the impact of hypercapnia on regulation of cellular cholesterol levels, and resultant effects on expression of ACE2 and entry of Pseudo-SARS-CoV-2 in cultured HBE, BEAS-2B and VERO cells, and airway epithelium of mice. Methods: Differentiated HBE, BEAS-2B or VERO cells were pre-incubated in normocapnia (5% CO2, PCO2 36 mmHg) or hypercapnia (15% CO2, PCO2 108 mmHg), both with normoxia, for 4 days. Expression of ACE2 and sterol regulatory element binding protein 2 (SREPB2), the master regulator of cholesterol synthesis, was assessed by immunoblot or immunofluorescence. Cholesterol was measured in cell lysates by Amplex red assay. Cells cultured in normocapnia or hypercapnia were also infected with Pseudo SARS-CoV-2, a Neon Green reporter baculovirus. For in vivo studies, C57BL/6 mice were exposed to normoxic hypercapnia (10% CO2/21% O2) for 7 days, or air as control, and airway epithelial expression of ACE2, SREBP2, ABCA1, ABCG1 and HMGCS1 was assessed by immunofluorescence. SREBP2 was blocked using the small molecules betulin or AM580, and cellular cholesterol was disrupted using MβCD. Results: Hypercapnia increased expression and activation of SREBP2 and decreased expression of ABC transporters, thereby augmenting epithelial cholesterol levels. Elevated CO2 also augmented ACE2 expression and Pseudo-SARSCoV- 2 entry into epithelial cells in vitro and in vivo. These effects were all reversed by blocking SREBP2 or disrupting cellular cholesterol. Conclusion: Hypercapnia augments cellular cholesterol levels by altering expression of cholesterol biosynthetic enzymes and efflux transporters, leading to increased epithelial expression of ACE2 and entry of Pseudo-SARS-CoV-2 into cells. These findings suggest that ventilatory support to limit hypercapnia or pharmacologic interventions to decrease cellular cholesterol might reduce viral burden and improve clinical outcomes of SARSCoV- 2 infection in advanced COPD and other severe lung diseases.

Clinical Characteristics and Outcomes of Patients With COVID-19-Associated Acute Respiratory Distress Syndrome Who Underwent Lung Transplant.

Importance: Lung transplantation is a potentially lifesaving treatment for patients who are critically ill due to COVID-19-associated acute respiratory distress syndrome (ARDS), but there is limited information about the long-term outcome. Objective: To report the clinical characteristics and outcomes of patients who had COVID-19-associated ARDS and underwent a lung transplant at a single US hospital. Design, Setting, and Participants: Retrospective case series of 102 consecutive patients who underwent a lung transplant at Northwestern University Medical Center in Chicago, Illinois, between January 21, 2020, and September 30, 2021, including 30 patients who had COVID-19-associated ARDS. The date of final follow-up was November 15, 2021. Exposures: Lung transplant. Main Outcomes and Measures: Demographic, clinical, laboratory, and treatment data were collected and analyzed. Outcomes of lung transplant, including postoperative complications, intensive care unit and hospital length of stay, and survival, were recorded. Results: Among the 102 lung transplant recipients, 30 patients (median age, 53 years [range, 27 to 62]; 13 women [43%]) had COVID-19-associated ARDS and 72 patients (median age, 62 years [range, 22 to 74]; 32 women [44%]) had chronic end-stage lung disease without COVID-19. For lung transplant recipients with COVID-19 compared with those without COVID-19, the median lung allocation scores were 85.8 vs 46.7, the median time on the lung transplant waitlist was 11.5 vs 15 days, and preoperative venovenous extracorporeal membrane oxygenation (ECMO) was used in 56.7% vs 1.4%, respectively. During transplant, patients who had COVID-19-associated ARDS received transfusion of a median of 6.5 units of packed red blood cells vs 0 in those without COVID-19, 96.7% vs 62.5% underwent intraoperative venoarterial ECMO, and the median operative time was 8.5 vs 7.4 hours, respectively. Postoperatively, the rates of primary graft dysfunction (grades 1 to 3) within 72 hours were 70% in the COVID-19 cohort vs 20.8% in those without COVID-19, the median time receiving invasive mechanical ventilation was 6.5 vs 2.0 days, the median duration of intensive care unit stay was 18 vs 9 days, the median post-lung transplant hospitalization duration was 28.5 vs 16 days, and 13.3% vs 5.5% required permanent hemodialysis, respectively. None of the lung transplant recipients who had COVID-19-associated ARDS demonstrated antibody-mediated rejection compared with 12.5% in those without COVID-19. At follow-up, all 30 lung transplant recipients who had COVID-19-associated ARDS were alive (median follow-up, 351 days [IQR, 176-555] after transplant) vs 60 patients (83%) who were alive in the non-COVID-19 cohort (median follow-up, 488 days [IQR, 368-570] after lung transplant). Conclusions and Relevance: In this single-center case series of 102 consecutive patients who underwent a lung transplant between January 21, 2020, and September 30, 2021, survival was 100% in the 30 patients who had COVID-19-associated ARDS as of November 15, 2021.

Early outcomes after lung transplantation for severe COVID-19: a series of the first consecutive cases from four countries.

BACKGROUND: Lung transplantation is a life-saving treatment for patients with end-stage lung disease; however, it is infrequently considered for patients with acute respiratory distress syndrome (ARDS) attributable to infectious causes. We aimed to describe the course of disease and early post-transplantation outcomes in critically ill patients with COVID-19 who failed to show lung recovery despite optimal medical management and were deemed to be at imminent risk of dying due to pulmonary complications. METHODS: We established a multi-institutional case series that included the first consecutive transplants for severe COVID-19-associated ARDS known to us in the USA, Italy, Austria, and India. De-identified data from participating centres-including information relating to patient demographics and pre-COVID-19 characteristics, pretransplantation disease course, perioperative challenges, pathology of explanted lungs, and post-transplantation outcomes-were collected by Northwestern University (Chicago, IL, USA) and analysed. FINDINGS: Between May 1 and Sept 30, 2020, 12 patients with COVID-19-associated ARDS underwent bilateral lung transplantation at six high-volume transplant centres in the USA (eight recipients at three centres), Italy (two recipients at one centre), Austria (one recipient), and India (one recipient). The median age of recipients was 48 years (IQR 41-51); three of the 12 patients were female. Chest imaging before transplantation showed severe lung damage that did not improve despite prolonged mechanical ventilation and extracorporeal membrane oxygenation. The lung transplant procedure was technically challenging, with severe pleural adhesions, hilar lymphadenopathy, and increased intraoperative transfusion requirements. Pathology of the explanted lungs showed extensive, ongoing acute lung injury with features of lung fibrosis. There was no recurrence of SARS-CoV-2 in the allografts. All patients with COVID-19 could be weaned off extracorporeal support and showed short-term survival similar to that of transplant recipients without COVID-19. INTERPRETATION: The findings from our report show that lung transplantation is the only option for survival in some patients with severe, unresolving COVID-19-associated ARDS, and that the procedure can be done successfully, with good early post-transplantation outcomes, in carefully selected patients. FUNDING: National Institutes of Health. VIDEO ABSTRACT.

Outcomes after extracorporeal membrane oxygenation support in COVID-19 and non-COVID-19 patients.

BACKGROUND: Veno-venous extracorporeal membrane oxygenation (V-V ECMO) support is increasingly used in the management of COVID-19-related acute respiratory distress syndrome (ARDS). However, the clinical decision-making to initiate V-V ECMO for severe COVID-19 still remains unclear. In order to determine the optimal timing and patient selection, we investigated the outcomes of both COVID-19 and non-COVID-19 patients undergoing V-V ECMO support. METHODS: Overall, 138 patients were included in this study. Patients were stratified into two cohorts: those with COVID-19 and non-COVID-19 ARDS. RESULTS: The survival in patients with COVID-19 was statistically similar to non-COVID-19 patients (p = .16). However, the COVID-19 group demonstrated higher rates of bleeding (p = .03) and thrombotic complications (p < .001). The duration of V-V ECMO support was longer in COVID-19 patients compared to non-COVID-19 patients (29.0 ± 27.5 vs 15.9 ± 19.6 days, p < .01). Most notably, in contrast to the non-COVID-19 group, we found that COVID-19 patients who had been on a ventilator for longer than 7 days prior to ECMO had 100% mortality without a lung transplant. CONCLUSIONS: These findings suggest that COVID-19-associated ARDS was not associated with a higher post-ECMO mortality than non-COVID-19-associated ARDS patients, despite longer duration of extracorporeal support. Early initiation of V-V ECMO is important for improved ECMO outcomes in COVID-19 ARDS patients. Since late initiation of ECMO was associated with extremely high mortality related to lack of pulmonary recovery, it should be used judiciously or as a bridge to lung transplantation.

Bacterial Superinfection Pneumonia in Patients Mechanically Ventilated for COVID-19 Pneumonia.

Rationale: Current guidelines recommend patients with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pneumonia receive empirical antibiotics for suspected bacterial superinfection on the basis of weak evidence. Rates of ventilator-associated pneumonia (VAP) in clinical trials of patients with SARS-CoV-2 pneumonia are unexpectedly low. Objectives: We conducted an observational single-center study to determine the prevalence and etiology of bacterial superinfection at the time of initial intubation and the incidence and etiology of subsequent bacterial VAP in patients with severe SARS-CoV-2 pneumonia. Methods: Bronchoscopic BAL fluid samples from all patients with SARS-CoV-2 pneumonia requiring mechanical ventilation were analyzed using quantitative cultures and a multiplex PCR panel. Actual antibiotic use was compared with guideline-recommended therapy. Measurements and Main Results: We analyzed 386 BAL samples from 179 patients with SARS-CoV-2 pneumonia requiring mechanical ventilation. Bacterial superinfection within 48 hours of intubation was detected in 21% of patients. Seventy-two patients (44.4%) developed at least one VAP episode (VAP incidence rate = 45.2/1,000 ventilator days); 15 (20.8%) initial VAPs were caused by difficult-to-treat pathogens. The clinical criteria did not distinguish between patients with or without bacterial superinfection. BAL-based management was associated with significantly reduced antibiotic use compared with guideline recommendations. Conclusions: In patients with SARS-CoV-2 pneumonia requiring mechanical ventilation, bacterial superinfection at the time of intubation occurs in <25% of patients. Guideline-based empirical antibiotic management at the time of intubation results in antibiotic overuse. Bacterial VAP developed in 44% of patients and could not be accurately identified in the absence of microbiologic analysis of BAL fluid.

Distinctive features of severe SARS-CoV-2 pneumonia.

The coronavirus disease 2019 (COVID-19) pandemic is among the most important public health crises of our generation. Despite the promise of prevention offered by effective vaccines, patients with severe COVID-19 will continue to populate hospitals and intensive care units for the foreseeable future. The most common clinical presentation of severe COVID-19 is hypoxemia and respiratory failure, typical of the acute respiratory distress syndrome (ARDS). Whether the clinical features and pathobiology of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pneumonia differ from those of pneumonia secondary to other pathogens is unclear. This uncertainty has created variability in the application of historically proven therapies for ARDS to patients with COVID-19. We review the available literature and find many similarities between patients with ARDS from pneumonia attributable to SARS-CoV-2 versus other respiratory pathogens. A notable exception is the long duration of illness among patients with COVID-19, which could result from its unique pathobiology. Available data support the use of care pathways and therapies proven effective for patients with ARDS, while pointing to unique features that might be therapeutically targeted for patients with severe SARS-CoV-2 pneumonia.

Hypercapnia Increases ACE2 Protein Expression and Pseudo-SARS-CoV-2 Virus Entry in Airway Epithelial Cells

Rationale: Individuals with chronic obstructive pulmonary disease (COPD) who develop 2019 coronavirus disease (COVID-19) are at increased risk of hospitalization, intensive care unit admission and death. COPD is associated with increased airway epithelial expression of angiotensin converting enzyme 2 (ACE2), the cell surface receptor to which the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein binds and which mediates entry of the virus into cells. Hypercapnia, the elevation of CO2 in blood and tissue, commonly develops in advanced COPD and is associated with frequent and potentially fatal pulmonary infections. We previously showed that normoxic hypercapnia alters expression of innate immune genes, including multiple viral response genes, in primary human bronchial epithelial (HBE) cells (Sci Reports 8:13508, 2018). Thus, in the current study, we explored the effect of hypercapnia on expression of ACE2 and uptake of a Pseudo-SARS-CoV-2 baculovirus by airway epithelial cells. Methods: HBE cells (Lonza) differentiated at air-liquid interface or immortalized BEAS-2B cells were pre-incubated in normocapnia (NC, 5% CO2, PCO2 36 mmHg) or normoxic hypercapnia (HC, 15% CO2, PCO2 108 mmHg) for 4 days. ACE2 protein expression was assessed by immunoblot or immunofluorescence (IF). In addition, BEAS-2B cells pre-exposed to NC or HC for 2 days were infected with Pseudo SARS-CoV-2 for an additional 2 days. Pseudo SARS-CoV-2 (Montana Molecular) is a reporter baculovirus whose surface is decorated with SARS-CoV-2 spike protein, and which induces expression of Neon Green protein in the nucleus of host cells 24 h after viral entry. For in vivo studies, C57BL/6 mice were pre-exposed to normoxic hypercapnia (10% CO2/21% O2) for 7 days, or air as control, and ACE2 expression in lung tissue was assessed by IF. Results: Compared to culture in NC, HC increased ACE2 protein expression by ∼4-fold in HBE cells and ∼2.5-fold in BEAS-2B cells. Likewise exposure of mice for 7 days to 10% CO2, as compared to air, markedly increased airway epithelial cell expression of ACE2 (Figure 1). Additionally, culture in HC, as compared to NC, increased Pseudo SARS-CoV-2 entry to BEAS-2B cells. Conclusion: Elevated CO2 increases airway epithelial cell expression of the SARS-CoV-2 receptor, ACE2, in vitro and in vivo. This may lead to a greater burden of SARS-CoV-2 infection in patients with hypercapnia, and in part account for worse clinical outcomes of COVID-19 pneumonia in advanced COPD and other severe lung diseases.

Computed tomography scan abnormalities in patients with post-acute COVID syndrome

Rationale: Patients recovering from COVID-19 infection can have persistent respiratory symptoms. These symptoms are part of a syndrome of prolonged recovery from of COVID-19 which has been termed 'Post-Acute Covid Syndrome (PACS).' Many patients with PACS have been found to have persistent radiographic changes. It is not known whether these radiographic changes represent developing fibrosis, a developing inflammatory process such as organizing pneumonia, or both. In this study we sought to characterize the radiographic changes seen in patients with persistent pulmonary symptoms. Methods: The medical records of patients who presented to the pulmonary clinic at the Comprehensive COVID Center were reviewed. Computed tomography (CT) scans were reviewed if obtained greater than 4 weeks after COVID diagnosis. If multiple CT scans were performed, the most recent scan was included. Radiographic abnormalities were categorized as inflammatory (ground-glass opacities or consolidation), fibrotic (traction bronchiectasis, reticulation, or honeycombing), both, or neither. Results: 33 patients were evaluated. During the acute phase of COVID infection 8 (24%) were admitted to the intensive care unit, 7 (21%) required mechanical ventilation, and 12 (36%) were admitted to the floor. 20 (61%) patients had CT that fit inclusion criteria. Of the 20 CT scans reviewed, 13 (65%) were abnormal. 10 (50%) scans demonstrated evidence of fibrosis, 11 (55%) scans demonstrated inflammatory changes, and 8 (40%) scans demonstrated both fibrosis and inflammatory changes. The average time from COVID diagnosis to recovery CT was 131 days. The average time from COVID diagnosis to scans with signs of fibrosis was 151 days, whereas the average time from COVID diagnosis to scans with inflammatory changes was 127 days. Conclusion: The etiology of persistent dyspnea in PACS is an area of active investigation, and radiographic patterns of injury may suggest underlying pathologic processes. Our study demonstrates abnormal radiographic findings, including evidence of both fibrotic and inflammatory parenchymal changes, in the majority of patients seen with PACS followed greater than 4 months after initial diagnosis. It is unknown if the ground glass opacities or consolidative changes are representative of post infectious organizing injury or fibrosis that is below the level of detection of CT resolution. Further prospective observational studies are warranted to determine if these changes are progressive, or if interventions such as steroids can expedite respiratory symptom recovery in the setting of a post-acute COVID clinic.

Comparing ecmo characteristics and outcomes among COVID-19 and non-COVID-19 patients at a tertiary academic medical center

Rationale: Coronavirus disease 2019 (COVID-19) can cause severe respiratory failure that worsens despite maximal medical management. When to initiate extracorporeal membrane oxygenation (ECMO) and how to manage these patients on ECMO is not clear. Here, we present our experience with venovenous ECMO to support patients with COVID-19 and compare it to historic patients supported with VV-ECMO for other causes of respiratory failure. Methods: Patients admitted to our tertiary academic medical center in 2019 and 2020 who received VV ECMO support were included in this retrospective chart review. We examined patients with and without COVID-19 infection. We placed COVID-19 patients on ECMO who failed supportive care with mechanical ventilation using a high PEEP low tidal volume strategy, prone positioning, and neuromuscular blockade. Data analysis were done in Excel and Prism. Non-parametric data were compared with unpaired, two-tailed Mann-Whitney tests. Results: ECMO was provided to 26 COVID-19 patients and 38 patients without COVID-19. Median (interquartile range) age of COVID-19 patients was 49.5 (40.5-56.25), compared with non-COVID-19 patients: 53.5 (30.5-60.25), p=0.28. COVID-19 patients had a significantly higher BMI: 32 (30.1-35.9) vs. 26.4 (23.6-29.4), p<0.001. There were 27% female COVID-19 patients compared with 37% female non-COVID patients (p=0.43). COVID-19 patients had similar PaO2:FiO2 ratios as non-COVID patients on day of cannulation: 74 (69-112) vs 78 (60-205), p=0.65. COVID-19 patients had longer ventilator duration pre-cannulation (not including time spent intubated at outside hospitals prior to transfer to our center)-1.9 (1.4-7.0) days vs 0.7 (-.2-1.0) days, p<0.001. COVID patients spent more days on ECMO compared with non-COVID patients: 20.7 (7.3-36.5) vs. 11.5 (3.8-26.8), p=0.14. Twelve (46%) of the COVID-19 ECMO patients died, compared with 9 (25%) of the non-COVID ECMO patients, p=0.10. Conclusions: In patients with severe SARS-CoV-2 pneumonia induced ARDS who fail maximal supportive therapy with mechanical ventilation, outcomes are similar or worse than patients historically receiving VV ECMO support for respiratory failure. These findings highlight the need to determine the optimal timing of ECMO initiation and management in patients with severe SARS-CoV-2 pneumonia.