Sars-Cov-2 Infection Promotes Endothelial Dysfunction and Thrombosis in a Mouse Model of COVID-19
Authors: Aiolfi Roberto , Ahmed Jeanine , de la Torre Juan Carlos , Griffin John H. , Mosnier Laurent O. 
Published: November 2021

Abstract
Coronavirus disease 2019 (COVID-19) is caused by infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Most infected individuals are asymptomatic or show only mild symptoms, but 20% of infected individuals become severely ill resulting in a 2-5% mortality rate for severe infections. Men, the elderly and patients with comorbidities (such as cardiovascular disease, hypertension, diabetes, and obesity) are more likely to develop severe disease. Clinical features characterizing severe COVID-19 cases include inflammation and thrombosis, but the molecular mechanisms underlying these processes remain elusive. K18 hACE2 transgenic mice express the SARSCoV-2 receptor human angiotensin-converting enzyme 2 (hACE2) under the control of the human cytokeratin 18 (K18) promoter. K18 hACE2 mice express hACE2 in airway epithelial cells and are susceptible to SARS-CoV-1 and SARS-CoV-2 infections. At the dose of 10 5 PFU/mouse, all SARS-CoV-2-infected K18 hACE2 mice rapidly lose weight and succumb to viral infection by 5-6 days post infection. Morbidity and mortality correlated with SARS-CoV-2 replication in the nasal turbinates and lungs. Notably, susceptibility was highly associated with a local and systemic cytokine/chemokine storm. SARS-CoV-2 infection in K18 hACE2 mice recapitulates many of the pathological findings observed in human patients offering a reliable animal model for the study of SARS-CoV-2 pathogenesis. Infection with a lower viral dose (10 4 and 2.5x10 3 PFU/mouse) prolongs the symptomatic phase of the infection, postponing time of death up to 16 days post infection (mortality rate at 10 4 PFU: ~40% in females, 100% in males; mortality rate at 2.5x10 3 PFU: ~30% in females, ~55% in males). At these lower viral doses, K18 hACE2 mouse males exhibited both increased susceptibility to the SARS-CoV-2 infection and more severe disease. Male mice showed increased mortality associated with an increase in weight loss and decrease in body temperature. Disease characteristics showed striking similarities with reported human COVID-19 cases, including severely reduced O 2 saturation. The pathogenesis of severe COVID-19 cases involves both virus-induced cell damage and secondary tissue damage due to a vicious cycle of dysregulated - hyperactive coagulation and inflammatory pathways that present as "a cytokine storm", endothelial dysfunction, and "immunothrombosis". Analysis of murine plasma analytes from infected mice revealed additional pathogenetic features resembling SARS-CoV-2 infection in humans. High circulating D-dimer levels are now considered a main predictor of poor outcome of SARS-CoV-2 infection. Notably, we also observed a progressive increase of circulating D-dimer levels in the plasma of K18 hACE2 infected mice peaking at day 7 post infection, suggestive of a hypercoagulable state. Moreover, similar to humans, the increase in soluble thrombomodulin plasma concentration and its correlation with disease severity was indicative of endothelial activation and dysfunction in K18 hACE2 infected mice. SARS-CoV-2 infection-induced changes of coagulation and endothelial activation in mice resulted in a biphasic alteration of endothelial permeability where an initial increase in vascular permeability, peaking at day 5 post infection, was followed by a sudden decrease in Evan's blue dye extravasation in the lung parenchyma and characterized by the appearance of areas of hemorrhagic infarction indicative of thrombotic events. Altogether, our results identify the K18 hACE2 transgenic mouse as an important small animal model to study the molecular mechanisms involved in the derangement of the finely tuned interaction between the immune and coagulation systems associated with severe cases of SARS-CoV-2 infections.

Disclosures
Mosnier: Hematherix: Membership on an entity's Board of Directors or advisory committees; Coagulant Therapeutics: Research Funding.