Poster session

[P1-2-44] Analysis of SARS-CoV-2 uptake mechanism in vascular endothelial cells

〇Yuya Sakurai^1,2, Nako Maishi¹, Aya Matsuda¹, Kyoko Hida¹ (1. Dept Vasc Biol Mol Pathol, Hokkaido Univ Grad Sch Dent Med, 2. Dept Dent Anesthesiol, Hokkaido Univ Grad Sch Dent Med)

Keywords:COVID-19、SARS-CoV-2、血管内皮細胞

[Background]Severe COVID-19 is characterized by the presence of vascular endothelial dysfunction as thrombosis and vasculitis. Although SARS-CoV-2 infection of vascular endothelial cells (ECs) has been considered, the presence or absence of viral infection of ECs remains controversial. One of the reasons for this is that the mechanism of viral uptake is not clear in ECs due to their low expression of ACE2. [Purpose]To identify molecules involved in viral uptake into ECs and to elucidate the detailed molecular mechanism. [Methods and Results]Young and mid-aged mice were intranasally inoculated with mouse-adapted SARS-CoV-2. Only the infected mid-aged mice showed marked weight loss and the histopathological findings in the lungs resembled those of severe COVID-19 lungs. Therefore, we isolated ECs from lungs of young and mid-aged mice as models of non-severe and severe COVID-19 infection and performed RNA-seq. Virus gene levels in lung ECs were significantly higher in mid-aged mice, but expression of ACE2 and TMPRSS2 was hardly detected. On the other hand, the expression of virus-responsive genes such as IRF7 and RIG-I was upregulated in mid-aged ECs, suggesting their virus uptake. Therefore, we considered the possibility of viral infection into ECs by the endocytosis pathway rather than the membrane fusion pathway. We further analyzed the molecular mechanism. We used siRNA to knock down viral receptors that had been reported to be expressed in other cell types and were expressed in ECs. We confirmed that the viral level was decreased, suggesting that these receptors function as viral receptors in ECs. We treated ECs with endocytosis inhibitor X to examine the involvement of the endocytosis pathway. We confirmed that the viral level was decreased, suggesting that certain pathway of endocytosis was involved in viral uptake into ECs. We are currently trying to investigate the molecular mechanism in more detail in the future and elucidate the relationship between SARS-CoV-2 uptake into ECs and COVID-19 severity.