Endothelial Cell Activation by SARS-CoV-2 Spike S1 Protein: A Crosstalk between Endothelium and Innate Immune Cells

Abstract

Background. Emerging evidences suggest that in severe COVID-19, multi-organ failure is associated with a hyperinflammatory state (the so-called “cytokine storm”) in combination with the development of a prothrombotic state. The central role of endothelial dysfunction in the pathogenesis of the disease is to date accepted, but the precise mechanisms underlying the associated coagulopathy remain unclear. Whether the alterations in vascular homeostasis directly depend upon the SARS-CoV-2 infection of endothelial cells or, rather, occur secondarily to the activation of the inflammatory response is still a matter of debate. Here, we address the effect of the SARS-CoV-2 spike S1 protein on the activation of human lung microvascular endothelial cells (HLMVEC). In particular, the existence of an endothelium-macrophage crosstalk in the response to the spike protein has been explored. Methods and Results. The effect of the spike protein is addressed in human lung microvascular endothelial cells (HLMVEC), either directly or after incubation with a conditioned medium (CM) of human monocyte-derived macrophages (MDM) previously activated by the spike S1 protein (CM-MDM). Both MDM and HLMVEC are activated in response to the S1 protein, with an increased expression of pro-inflammatory mediators. However, when HLMVEC are exposed to CM-MDM, an enhanced cell activation occurs in terms of the expression of adhesion molecules, pro-coagulant markers, and chemokines. Under this experimental condition, ICAM-1 and VCAM-1, the chemokines CXCL8/IL-8, CCL2/MCP1, and CXCL10/IP-10 as well as the protein tissue factor (TF) are markedly induced. Instead, a decrease of thrombomodulin (THBD) is observed. Conclusion. Our data suggest that pro-inflammatory mediators released by spike-activated macrophages amplify the activation of endothelial cells, likely contributing to the impairment of vascular integrity and to the development of a pro-coagulative endothelium.

Immune-Mediated Inflammatory Responses of Alveolar Epithelial Cells: Implications for COVID-19 Lung Pathology

“…To date, however, the pathogenic mechanisms underlying the hyperinflammatory response that contributes to alveolar epithelial injury remain incompletely understood. Recently, we and others demonstrated that the in vitro treatment of human macrophages with S1 of SARS-CoV-2 causes a massive release of cytokine and chemokines in the extracellular medium [17][18][19][20]; this, in turn, results in a significant strengthening of endothelial responses to the viral protein [17], sustaining the contribution of both direct and immune-mediated mechanisms in endothelial cell activation in COVID-19. In this context, the aim of the present study was to define the cellular and molecular pathways underlying the response of alveolar epithelial cells to either the spike S1 protein or to a mixture of inflammatory mediators secreted by S1-activated macrophages, so as to help clarify the pathophysiology of lung disease upon SARS-CoV-2 infection.…”

Role of low-dose tissue plasminogen activator in patients with refractory hypoxia due to presumed microthrombi in pulmonary vasculature in coronavirus disease 2019: A case series and review of the literature

“…Therefore, a combination of endothelial damage and inflammation leads to a hypercoagulable state that leads to the generation of microthrombi. [ 15 ] The vast and unexplored spectrum of inflammation, endothelial injury, and pulmonary microcirculatory thrombosis contributes markedly to COVID-19-related ARDS. [ 16 ] Various conditions predispose COVID-19 patients to bleeding risk disseminated intravascular coagulation, thrombocytopenia, COVID-related hemophagocytosis, complications, and overuse of anticoagulants.…”

Desmopressin Stimulates Nitric Oxide Production in Human Lung Microvascular Endothelial Cells

“…mRNA expression has been analyzed through RT-qPCR, as previously described [11], upon incubation of HLMVEC with 1 and 100 nM dDAVP for 1 or 16 h. 1 µg of cDNA was obtained through the reverse transcription of total RNA with the RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, Milano, Italy). qPCR was then performed on a StepOnePlus Real-Time PCR System (Thermo Fisher Scientific) by employing specific forward/reverse primer pairs (Table 1) and SYBR™ Green or TaqMan Gene Expression Master Mix (Thermo Fisher Scientific).…”

“…The expression level of genes coding for vasopressin receptors (AVPR1A and AVPR2) was determined using the formula 2∆Ct (where ∆Ct = Ct RPL15 − Ct gene of interest ) [12], after normalization for the housekeeping gene (Ribosomal like protein 15, RPL15, Gene ID: 6138). The amount of the different genes upon treatment with dDAVP was, instead, calculated with the ∆∆Ct method [11] and expressed, relatively to RPL15, as fold change of control untreated cells (=1).…”