SARS-CoV-2 Spike Protein Induces Paracrine Senescence and Leukocyte Adhesion in Endothelial Cells

Abstract

Increased mortality in COVID-19 often associates with microvascular complications. We have recently shown that SARS-CoV-2 spike protein promotes an inflammatory cytokine IL-6/IL-6R induced trans-signaling response and alarmin secretion. Virus infected or spike transfected human epithelial cells exhibited an increase in senescence state with the release of senescence associated secretory proteins (SASP) related inflammatory molecules. Introduction of BRD4 inhibitor AZD5153 to senescent epithelial cells reversed this effect and reduced SASP related inflammatory molecule release in TMNK-1 or EA hy926 as representative human endothelial cell line, when exposed to cell culture medium (CM) derived from A549 cells expressing SARS-CoV-2 spike protein, also exhibited a senescence phenotype with enhanced p16, p21, SA-β-galactosidase expression, and triggered SASP pathways. Inhibition of IL-6 trans-signaling by Tocilizumab and inhibition of inflammatory receptor signaling by the BTK inhibitor Zanubrutinib, prior to exposure of CM to endothelial cells, inhibited p21 and p16 induction. We also observed an increase in reactive oxygen species (ROS) in A549 spike transfected and endothelial cells exposed to spike transfected CM. ROS generation in endothelial cell lines was reduced after treatment with Tocilizumab and Zanubrutinib. Cellular senescence was associated with an increased level of the endothelial adhesion molecules, VCAM-1 and ICAM-1 with in vitro leukocyte attachment potential. Inhibition of senescence or SASP function prevented VCAM-1/ICAM-1 expression and leukocyte attachment. Taken together, we identified that the exposure of human endothelial cells to cell culture supernatant derived from SARS-CoV-2 spike protein expression displayed cellular senescence markers, leading to enhanced leukocyte adhesion. Importance: The present study was aimed at examining the underlying mechanism of extrapulmonary manifestations of SARS-CoV-2 spike protein associated pathogenesis, with the notion that infection of the pulmonary epithelium can lead to mediators that drive endothelial dysfunction. We utilized SARS-CoV-2 spike protein expression in cultured cells of human hepatocytes (Huh7.5) and pneumocytes (A549) to generate conditioned culture media (CM). Endothelial cell lines (TMNK-1 or EA hy926 ) treated with CM exhibited increase in cellular senescence markers by a paracrine mode, and lead to leukocyte adhesion. Overall, the link between these responses in endothelial cell senescence, and a potential contribution to microvascular complication in productively SARS-CoV-2 infected humans is implicated. Furthermore, the use of inhibitors (BTK, IL-6 and BRD4) showed reverse effect in the senescent cells. These results may support the selection of potential adjunct therapeutic modalities to impede SARS-CoV-2 associated pathogenesis.

Pulmonary infection by SARS-CoV-2 induces senescence accompanied by an inflammatory phenotype in severe COVID-19: possible implications for viral mutagenesis

“…In line with this notion, it has been previously reported in vitro that cellular senescence has also a role as a natural antiviral defence mechanism and that SASP acts as the major contributor of this response, activating and recruiting the immune system to clear out the infection [ 21 , 22 ]. Subsequently, other cellular compartments (stem/progenitor, endothelial and inflammatory cells) may be affected via different cell and non-cell autonomous mechanisms, leading to aberrant immune responses, chronic inflammation, tissue dysfunction and/or fibrosis and eventually to lung damage and failure [ 26 , 55 ]. In particular, senescence in stem/progenitor cells impairs lung regenerative capacity.…”

SARS-CoV-2 Spike Protein S1-Mediated Endothelial Injury and Pro-Inflammatory State Is Amplified by Dihydrotestosterone and Prevented by Mineralocorticoid Antagonism

“…We employed the functionally active subunit of spike protein S1 of SARS-CoV2 that has shown binding to ACE2 in a cellular model, and this binding was abolished by the co-administration of receptor-binding domain (RBD) or neutralizing antibody to S1 [28]. The spike protein subunit S1 of SARS-CoV2 has previously shown to upregulate intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) protein expression in human umbilical vein endothelial cells [29], and caused permeability and barrier dysfunction in pulmonary endothelial cells in vitro [30]. Spike protein shedding of SARS-CoV-2 from the surface of the virions and infected cells may play an important role in COVID-19 pathogenesis [31,32] and prior reports support that spike protein alone can elicit some of the disease manifestation in COVID-19 [33,34].…”

Identifying potential novel insights for COVID-19 pathogenesis and therapeutics using an integrated bioinformatics analysis of host transcriptome

“…Telomere dysfunction may provide a novel pathway for the pathogenesis of COVID-19. Homologous recombination (HR), non-homologous end-joining (NHEJ), and cellular senescence enriched for module 3 may play a vital role in COVID-19 pathogenesis [64] , [65] , [66] , [67] . NHEJ or HR machinery are involved in the interactions between viruses and the host DNA damage response (DDR) machinery [64] , [65] .…”

“…Lee S et al proposed that senolytic targeting of virus-infected cells is considered as a novel treatment option against SARS-CoV-2 [66] . Cellular senescence induced by virus is accompanied by a senescence-associated secretory phenotype (SASP) with increased pro-inflammatory cytokines, extracellular matrix-active factors and pro-coagulatory mediators [67] .…”