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.
ABSTRACT
Background: Coronavirus disease 2019 (COVID-19) caused by SARS-CoV-2 was first reported in Wuhan in 2019 and reached pandemic proportions. SARS-CoV-2-related respiratory failure and acute kidney injury (AKI) are major complications of infection. Kidney Injury Molecule-1 (KIM-1) is a scavenger receptor expressed by kidney epithelial cells and was previously reported to be a receptor for Hepatitis virus A. We hypothesized that KIM-1 is a receptor for SARS-CoV-2 and may play an important role in COVID-19 lung and kidney injury. Methods: Human lung and kidney autopsy samples were immunostained and analyzed. Liposomal nanoparticles displaying the SARS-CoV-2 spike protein on their surface (virosomes) were generated. Virosome uptake by A549 lung epithelial cells, mouse primary lung epithelial cells and human kidney tubuloids (3D structures of kidney epithelial cells) was evaluated in the presences or absence of anti-KIM-1 antibody or TW-37, a small molecule inhibitor of KIM-1-mediated endocytosis that we discovered. Protein-protein interaction characteristics between purified SARS-CoV-2 spike protein and purified KIM-1 were determined using microscale thermophoresis. HEK293 cells expressing human KIM-1 but not angiotensin-converting enzyme 2 (ACE2) were infected with live SARS-CoV-2 or pseudovirions expressing the SARS-CoV-2 spike protein. Results: KIM-1 was expressed in lung and kidney epithelial cells in COVID-19 patient autopsy samples. Human and mouse lung and kidney epithelial cells expressed KIM-1 and endocytosed spike-virosomes. Both anti-KIM-1 antibodies and TW-37 inhibited uptake. Enhanced KIM-1 expression by human kidney tubuloids increased virosome uptake. KIM-1 positive cells expressed less ACE2. Using microscale thermophoresis, the EC50 for interaction between KIM-1 and SARS-CoV-2 spike protein and the receptor binding domain were 56.2±28.8 nM and 9.95±3.10 nM, respectively. KIM-1-expressing HEK293 cells without ACE2 expression had increased susceptibility to infection by live SARS-CoV-2 and pseudovirions expressing spike when compared with control cells. Conclusions: KIM-1 is a receptor for SARS-CoV-2 in the lung and kidney and thus, KIM-1 inhibitors such as TW-37 can be potential therapeutics and/or prophylactic agents for COVID-19.
ABSTRACT
Objective: To report neuropathological findings and quantify SARS-CoV-2 viral burden for 18 consecutive coronavirus disease 2019 (COVID-19) autopsies. Background: COVID-19 is a respiratory disease caused by SARS-CoV-2, a virus known to infect lung epithelial cells, yet data about SARS-CoV-2 neuropathology in human brain autopsies is limited. Design/Methods: Brain tissue specimens were sampled from 18 subjects (10 standard areas), fixed in formalin, and stained with hematoxylin and eosin for histopathological analysis. SARSCoV-2 immunohistochemistry and reverse transcription quantitative polymerase chain reaction (RT-qPCR) were performed on 10 brain sections from 2 subjects and 2 sections (medulla and frontal lobe with olfactory nerve) from the remaining 16 subjects. Results: Median age was 62 years (interquartile range, 53 to 75), and 14 patients (78%) were men. Presenting neurologic symptoms were myalgia (n=3), headache (n=2), and decreased taste (n=1);11 received mechanical ventilation. Acute hypoxic injury was detected in cerebrum, hippocampus, and cerebellum in all patients;rare foci of perivascular lymphocytes (n=2) or focal leptomeningeal inflammation (n=1) were also detected. RT-qPCR showed limited evidence of viral RNA. In 10 unique specimens from two subjects, results were equivocal (viral load <5.0 copies/mm3) in 4 and 5 sections, respectively. In the remaining 16 patients, 3 medulla sections and 3 frontal lobe and olfactory sections were positive (5.0 to 59.4 copies/mm3) while the rest were equivocal or negative. SARS-CoV-2 viral load did not correlate with the interval between the onset of symptoms and death or histopathological findings. Immunohistochemical staining for SARS-CoV-2 nucleocapsid protein was negative in neurons, glia, endothelium, and immune cells. Conclusions: Histopathology of brain specimens revealed hypoxia with limited evidence of direct viral damage, including no viral protein. Concordantly, although SARS-CoV-2 was detected by RT-qPCR in some sections, viral load was low and did not correlate with other pathological features.