Cellular and Molecular Consequences of SARS-CoV2 Infection in Pulmonary Vascular Endothelium

Clinical Center

Investigators

Abstract

IIn FY21, we determined the expression levels of known and putative host viral entry molecules including angiotensin-converting enzyme 2 (ACE2), transmembrane serine protease 2 (TMPRSS2), neuropilin 1 (NRP1), and DC-SIGNR (CD209L/CLEC4M) in primary human lung endothelial cells (ECs). Results were compared to human embryonic kidney (HEK293) cells, a cell line that is known to be permissive to SARS-CoV-2 entry. Additionally, experiments utilizing both SARS-CoV-2 pseudotyped lentiviral particles as well as live SARS-CoV-2 (WA1/2020) were conducted in various types of primary human ECs and the human Ea.hy926 endothelial line in order to determine whether human ECs were susceptible to SARS-CoV-2 infection in vitro. The results of this work revealed that primary human pulmonary artery and lung microvascular ECs express low levels of ACE2, TMPRSS2, and CD209L but high levels of NRP1. Neither Wuhan variant pseudotyped nor live USA/WA1 variant SARS-CoV-2 virus appear to generally infect primary human pulmonary ECs in vitro. Furthermore, overexpression of TMPRSS2 with ACE2 in HEK293 cells further increased infection with SARS-CoV-2 pseudotyped lentivirus whereas overexpression of NRP1 with ACE2 or CD209L with ACE2+TMPRSS2 appears to decrease infection compared to ACE2 overexpression alone. In conclusion, low levels of ACE2 and TMPRSS2 expression in cultured primary human lung ECs may explain their diminished susceptibility to SARS-CoV-2 infection. Future work is planned to determine whether overexpression of ACE2 and/or TMPRSS2, cell matrix, circulating factors in COVID+ patient serum or plasma, or different spike protein variants impact human EC susceptibility to infection in vitro. In FY22, in vitro exposure to increasing concentrations of plasma from hospitalized patients with mild and severe cases of COVID-19 did not consistently increase or decrease ACE2 and NRP1 mRNA expression in primary human pulmonary artery ECs (PAECs). In contrast to wild-type Wuhan variant pseudotyped viral particles, SARS-CoV-2 pseudovirus expressing the D614G spike protein variant successfully entered PAECs (P<0.05 for MOI 2.5 and 5 compared to MOI 0; P<0.05 for the fold increase compared to the Wuhan strain at the same MOI), while alternative variants (N439K and N501Y) did not. Likewise, using immunofluorescent microscopy, recombinant spike protein from the SARS-CoV-2 Delta variant (contains D614G mutation) dose-dependently entered PAECs while the Wuhan variant recombinant spike protein did not. Yet following inoculation with either the USA/WA1 strain, an early pandemic isolate carrying the D614G mutation, or the Delta variant of SARS-CoV-2 variant, there was no evidence of viral replication as determined by viral quantification (TCID50) of PAEC supernatants. In summary, while SARS-CoV-2 pseudovirus entry into pulmonary artery endothelium is influenced by spike protein variant expression, SARS-CoV-2 virus does not appear to effectively replicate in primary human pulmonary ECs in vitro.