B65-03 ICU-Sleep Fragmentation Disrupts the Circadian Cycling of BMAL1 in Lung Endothelial Cells

Rationale Patients in the intensive care unit (ICU) have disrupted sleep patterns with reduced total sleep time and fragmentation of sleep. Mice subjected to ICU-sleep fragmentation (SF) demonstrated significantly delayed lung recovery after pneumonia, although the mechanism of how this occurs is unknown. The clock protein BMAL1 is a driver of the circadian rhythm and endothelial proliferation. We hypothesize that ICU-SF disrupts the circadian cycling of BMAL1 which may affect the proliferative potential of lung endothelial cells. Methods Mechanistic studies that examine the circadian influence on endothelial proliferation are challenging, because primary cells lose their circadian rhythm in culture. Therefore, we developed an approach to initiate the circadian rhythm of primary endothelial cells. To induce circadian cycling, human lung microvascular endothelial cells (hLMVECs) were kept in 1% FBS media overnight and then stimulated with 100nM of dexamethasone for 2 hours. BMAL1 mRNA levels were tracked using a BMAL1 promoter driven fluorescent reporter system (BMAL1:mScarlet), and protein expression was assessed with western blotting. In vivo, mice were subjected to a recently validated model of ICU-SF. After ICU-SF, lung endothelial cells were isolated and used for western blotting. Results Unstimulated hLMVECs displayed high BMAL1 mRNA and protein expression that did not fluctuate over time. After initiating rhythmicity, hLMVECs showed oscillation of BMAL1 mRNA and protein levels over 5 days with peak expression occurring in 24-hour intervals. The proliferative protein PCNA cycled over 24-hours, and its protein expression was inversely related to BMAL1 protein levels. In vivo, lung endothelial cells isolated from mice showed circadian cycling, having the highest BMAL1 protein expression at night (∼10:30 pm) and the lowest BMAL1 expression around noon (∼12:30 pm). In contrast, lung endothelial cells from ICU-SF exposed mice had a marked reduction in BMAL1 protein expression across all time points with little to no detectable protein at night. Conclusion Cultured hLMVECs do not have a circadian rhythm, whereas inducing rhythmicity shows cycling of proteins (BMAL1 and PCNA) that are important for proliferation. Therefore, initiating the circadian rhythm may provide a better in vitro model for studying pathways related to cell death and/or proliferation of cultured cells. In vivo, ICU-SF disrupts circadian cycling of BMAL1 in mouse lung endothelial cells. Future studies are needed to understand how ICU-SF disruption of the circadian rhythm affects endothelial proliferation during lung injury resolution.