ABSTRACT
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.
ABSTRACT
Introduction: We aimed to analyze the changes in depression, sleepiness, insomnia, and sleep habits in relation to the degree of self-isolation due to COVID-19 pandemic. A state of emergency was declared for the whole of Japan on 7 April 2020. People in Shiga prefecture were recommended to stay at home and refrain from moving to other prefectures from 8 April to 31 May 2020. Methods: We enrolled 54 patients who regularly visited the sleep outpatient clinic in Shiga University of Medical Science Hospital, Japan. We compared the sleep habits, depression (Patient Health Questionnaire-9: PHQ-9), insomnia (Athens Insomnia Scale: AIS), and sleepiness (Epworth Sleepiness Scale: ESS) of patients, one year before (from April to July 2019), during (May 2020) and six months after (Nov 2020) the self-isolation period due to the COVID-19. We conducted repeated measures ANOVA to examine changes. Results: PHQ-9 (3.89±0.64, 3.65±0.62 and 3.52±0.66: p=0.410), AIS (4.78±0.59, 4.65±0.57 and 4.63±0.57: p=0.843) and ESS (7.93±0.76, 7.07±0.68 and 7.00±0.73: p=0.088) were not significantly different among three visits. Sleep duration (6.06±0.23hr, 6.29±0.19hr and 6.16±0.22hr: p=0.248) and sleep onset latency (24.8±5.55min, 19.2±3.97min and 21.0±4.85min: p=0.445) were also not significantly different. Conclusion: Self-isolation due to COVID-19 was not associated with changes in depression, sleepiness, and insomnia among patients for sleep outpatient units in in Shiga prefecture, Japan.