Cellular and Molecular Consequences of SARS-VoV2 Infection in Pulmonary Vscular Endothelium

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Severe acute respiratory syndrome coronavirus-2 (SARS-CoV2), a novel coronavirus identified in December 2019, is now responsible for an expanding pandemic resulting so far in more than 300,000 deaths worldwide. While hypoxic respiratory failure due to SARS-CoV2 pneumonia is the most common presentation of severe illness, cardiovascular manifestations including shock, coagulopathy, venous thromboembolism, stroke and pulmonary hypertension (PH) appear to be major causes of mortality. Recent cases of critically ill children presenting with features of Kawasakis Disease and shock also highlight the potential of SARS-CoV2 infection to result in an acute or delayed syndrome characterized by widespread vascular inflammation. Importantly, autopsy studies in patients with coronavirus disease 2019 (COVID-19) demonstrated evidence of viral inclusions in endothelial cells, endothelial cell apoptosis, perivascular inflammatory infiltrates and microvascular thrombi in distal pulmonary arterioles. Furthermore, the first US case series of critically ill patients with COVID-19 reported that vasopressor-dependent shock developed in nearly all patients that required mechanical ventilation. Due to the absence of a bacterial co-infection in these patients, the authors implicated a direct, virally-mediated affect on the vasculature. Yet, a mechanistic understanding of these cardiovascular and pulmonary vascular complications is lacking. Our laboratory specializes in cellular models of endothelial dysfunction in the context of pulmonary arterial hypertension (PAH) (Awad KS and Elinoff JM et al. Am J Physiol Lung Cell Mol Physiol 2016). Pathologic vascular remodeling in PAH, while chronic rather than acute, is characterized by a proliferative, pro-thrombotic and inflammatory cellular phenotype. Notably, interferon driven-inflammation has been a prominent feature both in vivo and in some of our in vitro models of PAH-associated genetic defects. Our lab also has relevant expertise investigating the interface between the renin-angiotensin-aldosterone system and inflammation. We recently demonstrated that spironolactone broadly suppresses inflammatory signaling in human pulmonary endothelial cells by promoting the proteasomal degradation of the TFIIH subunit, XPB (Elinoff JM et al. Cardiovasc Res 2018). This previously unrecognized mineralocorticoid receptor-independent mechanism by which spironolactone suppresses vascular inflammation may be a useful adjunct for ameliorating the multisystem circulatory manifestations of COVID-19. Here, we plan to investigate the cellular, molecular and transcriptomic consequences of SARS-CoV-2 infection in human primary endothelial cells. In parallel studies, the impact of clinically relevant interventions and treatments, such as oxygen therapy, nitric oxide, ascorbic acid and renin-angiotensin-aldosterone system modulators including spironolactone will be investigated for their effects on SARS-CoV-2-induced endothelial cell dysfunction and injury. Specific Aims: 1. Infect primary human endothelial cells from clinically relevant vascular compartments and the human Ea.hy926 endothelial line with viable SARS-CoV2 and characterize the cellular phenotypic consequences. Circulating inflammatory mediators and markers of endothelial dysfunction in subjects with severe COVID-19 will be compared to conditioned media from endothelial cell monolayers infected with SARS-CoV2. 2. Examine the effects of SARS-CoV2 pseudotyped lentiviral particles and recombinant spike protein, as well as mammalian expression vectors encoding SARS-CoV2 genes, on human endothelial cells. 3. Test clinically relevant therapeutics for effects on SARS-CoV2 endothelial model systems.