ABSTRACT
Background: The pathobiology of in situ pulmonary thrombosis in acute respiratory distress syndrome (ARDS) due to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is incompletely characterized. In human pulmonary artery endothelial cells (HPAECs), hypoxia upregulates expression of a pro-thrombotic NEDD9 peptide (N9 ) on the extracellular plasma membrane surface. We hypothesized that increased pulmonary endothelial N9 is a novel feature of the SARS-CoV-2 pathophenotype. Methods: Paraffin-embedded autopsy lung specimens were acquired from patients with ARDS due to SARS-CoV-2 infection (n=13), ARDS of other causes (n=10), and non-disease controls (n=5). Immunofluorescence characterized expression of N9 , fibrin, and TCF12, a putative binding target of SARS-CoV-2 and known transcriptional regulator of NEDD9. We performed RNA-Seq on mRNA isolated from control HPAECs treated with normoxia or hypoxia (0.2% O2 ) for 24 hr. Immunoprecipitation-liquid chromatography-mass spectrometry (IP-LC-MS) profiled protein-protein interactions involving N9 relevant to thrombus stabilization. Results: Compared to non-SARS-CoV-2-ARDS lungs, pulmonary endothelial N9 expression and N9-fibrin colocalization was increased by 174% (P<0.002) and 212% (P<0.001) in SARS-CoV-2-ARDS, respectively. Compared to normoxia, hypoxia increased TCF12 mRNA quantity significantly in HPAECs in vitro [+1.19-fold, P=0.001;false discovery rate (FDR)=0.005]. Pulmonary endothelial nuclear TCF12 expression was also increased by 370% in SARS-CoV-2-ARDS vs. controls. In HPAEC plasma membranes, IP-LC-MS identified a novel protein-protein interaction between NEDD9 and the β3 subunit of the αvβ3 integrin, which regulates fibrin anchoring to endothelial cells. Conclusions: Compared to non-SARS-CoV-2-ARDS, SARS-CoV-2-ARDS is associated with increased pulmonary endothelial N9 expression and N9-fibrin colocalization in microthrombi in situ. Increased hypoxia signaling or SARS-CoV-2-mediated regulation of TCF12 are potential mechanisms by which to explain these findings. Identifying N9 in the pulmonary microthrombi of SARS-CoV-2 lungs may have important pathobiological and, potentially, therapeutic implications for ARDS patients.