A key role for endosomal NADPH oxidase in endothelial tissue factor expression
COVID-19 is associated with a high incidence of thrombotic complications.1 Necropsy studies reveal platelet-fibrin plugs in pulmonary arterioles, likely contributing to the hypoxaemia characteristic of advanced infection.2 It has been suggested that the thrombotic diathesis associated with COVID-19 reflects an endotheliopathy induced by viral infection of endothelial cells.3–5 These cells prominently express the ACE2 plasma membrane protein to which the spike protein of SARS-CoV-2 virions bind, enabling their endosomal incorporation into cells.6 7 The thrombotic complications of COVID-19 infection would be readily explained if SARS-CoV-2 infection of endothelial cells induces luminal expression of tissue factor (TF), which could then interact with circulating coagulation factor VII to trigger a proteolytic cascade culminating in the generation of thrombin and fibrin (extrinsic clotting). TF expression is negligible in healthy non-inflamed endothelial cells, but it can be upregulated at the transcription level by various proinflammatory stimuli that activate the NF-kappaB transcription factor. More specifically, the heterodimers p65/p50 or p65/c-Rel can bind to a novel ‘TF-kappaB’ sequence in the promoter of the TF gene, driving its induced expression.8–10
Various proinflammatory factors that induce TF in endothelial cells—including tumour necrosis factor-alpha (TNFα), antiphospholipid antibodies (aPL), ultrafine pollutant particles and homocysteine—have been shown to do so via signalling pathways in which activation of NADPH oxidase complexes plays an obligate role.11–14 The effects of aPL and of TNFα in this regard hinge on the activation of NADPH oxidase in endosomes which has incorporated these agonists.12
Prior to the emergence of SARS-CoV-2, it was demonstrated that a number of RNA viruses can activate endosomal NADPH oxidase through a mechanism dependent on toll-like receptor 7 (TLR7), which is activated by binding to single-stranded RNA.15 Presumably, these viruses, after binding to cellular plasma membranes, are incorporated into endosomes, and viral RNA released from the virions can interact with endosomal TLR7, triggering NADPH oxidase activation. Indeed, To and colleagues found that eight different types of single-stranded RNA viruses activated endosomal NADPH oxidase in alveolar macrophages, and the two types that did not activate it do not employ endosomes as their primary entry mechanism.15 Moreover, this effect was absent in alveolar macrophages in which TLR7 expression was knocked out.
SARS-CoV-2 is likewise a single-stranded RNA virus, the intracellular uptake of which is mediated by endosomes.16 We postulated that SARS-CoV-2, after incorporation into endosomes within endothelial cells, can likewise activate endosomal NADPH oxidase via TLR7, and that the resulting local production of superoxide/hydrogen peroxide leads to activation of NF-kappaB—by a mechanism yet to be determined—and subsequently to increased expression of TF. At present, while it is difficult to trace clinical studies that have measured serum markers of oxidative stress in COVID-19 patients, the fact that clinical outcomes were poorer in those provinces of China where soil selenium is deficient is compatible with the view that oxidant stress plays a key pathogenic role in this syndrome, and selenium is required for function of multiple antioxidant enzymes, including glutathione peroxidases and thioredoxin reductases.17 18 Moreover, oxidative stress is a key feature of other viral diseases that evoke acute respiratory distress syndrome and cytokine storm.19