Sustained Vascular Inflammatory Effects of SARS-CoV-2 Spike Protein on Human Endothelial Cells

Abstract

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has been associated with systemic inflammation and vascular injury, which contribute to the development of acute respiratory syndrome (ARDS) and the mortality of COVID-19 infection. Moreover, multiorgan complications due to persistent endothelial dysfunction have been suspected as the cause of post-acute sequelae of SARS-CoV-2 infection. Therefore, elucidation of the vascular inflammatory effect of SARS-CoV-2 will increase our understanding of how endothelial cells (ECs) contribute to the short- and long-term consequences of SARS-CoV-2 infection. Here, we investigated the interaction of SARS-CoV-2 spike protein with human ECs from aortic (HAoEC) and pulmonary microvascular (HPMC) origins, cultured under physiological flow conditions. We showed that the SARS-CoV-2 spike protein triggers prolonged expression of cell adhesion markers in both ECs, similar to the effect of TNF-α. SARS-CoV-2 spike treatment also led to the release of various cytokines and chemokines observed in severe COVID-19 patients. Moreover, increased binding of leucocytes to the endothelial surface and a procoagulant state of the endothelium were observed. Transcriptomic profiles of SARS-CoV-2 spike-activated HPMC and HAoEC showed prolonged upregulation of genes and pathways associated with responses to virus, cytokine-mediated signaling, pattern recognition, as well as complement and coagulation pathways. Our findings support experimental and clinical observations of the vascular consequences of SARS-CoV-2 infection and highlight the importance of EC protection as one of the strategies to mitigate the severe effects as well as the possible post-acute complications of COVID-19 disease.

Keywords: Endothelial cells; SARS-CoV-2; Spike protein; Vascular inflammation.

Fig. 1

SARS-CoV-2 spike protein-activated endothelial cells exhibit prolonged ICAM1 expression. Human pulmonary microvascular endothelial cells (HPMC) and human aortic endothelial cells (HAoEC) grown under flow conditions (10 dyne/cm²) and treated with 1 μg/ml SARS-CoV-2 spike protein, 1 ng/ml TNF-α, or remained untreated for 24 h. The cells were washed after the activation and further cultured for a total of 96 h post-activation. At the indicated time point, cells were fixed and stained for ICAM1 (green), E-Selectin (yellow), VE-Cadherin (white), and nuclei (blue) (a). Figures depict representative images. Quantification of the coverage of ICAM1 (b, c) as well as E-Selectin (d, e) at 24 h and 96 h post-treatment, respectively, were obtained from at least three biological replicates. Statistical analysis was done using one-way ANOVA with multiple comparisons

Fig. 2

SARS-CoV-2 spike triggers cytokine and chemokine releases from the endothelial cells. Expression of various cytokines and chemokines in human pulmonary microvascular cells (HPMC, a) and human aortic endothelial cells (HAoEC, b) were measured using a commercial bead-based multiplex ELISA and shown in log2 fold induction over mock at 24 h and 96 h post-treatment with 1 μg/ml SARS-CoV-2 spike protein or 1 ng/ml TNF-α

Fig. 3

SARS-CoV-2 spike activation leads to increased leucocyte adhesion to the surface of the endothelium. After 24 h activation with 1 μg/ml SARS-CoV-2 spike protein or 1 ng/ml TNF-α, HPMC and HAoEC were perfused with fluorescently labeled PBMCs for 20 min. Representative images from the time-series recording of PBMC adhesion to HPMC and HAoEC 24 h and 96 h post-treatment (a). Adhered PBMCs (defined as PBMCs that adhered for more than 3 s in the frame) to HPMC and HAoEC per 20 min at 24 h (b) and 96 h (c) post-treatment. Statistical analysis was done using one-way ANOVA with multiple comparisons

Fig. 4

SARS-CoV-2 spike induces a procoagulant state of the human endothelium. SARS-CoV-2 spike and TNF-α-treated HPMC and HAoEC were perfused with recalcified citrated human plasma spiked with fluorescently labeled fibrinogen. Representative images of clot formation HPMC and HAoEC 24 h and 96 h post-treatment (a). Time to clot formation was determined from the time series imaging, defined as the time when complete occlusion of the channel and the formation of saturated fluorescence signal from the fluorescently labeled fibrinogen were observed. Clotting time for both ECs at 24 h (b) and 96 h (c) after treatment. One-way ANOVA with multiple comparisons was used for statistical analysis

Fig. 5

Transcriptional change due to SARS-CoV-2 activation in the human endothelium. Venn diagram depicting overlapping differentially expressed genes (DEGs) for HPMC and HAoEC 24 h and 96 h post-treatment with SARS-CoV-2 spike and TNF-α (a). Heatmap of hierarchical clustering of all DEGs identified for HPMC (b) and HAoEC (c). Expression level was shown as the log-transformed of the average normalized counts from four independent replicates. Biological process enrichment analysis results for upregulated DEGs in HPMC (d, e) and HAoEC (f, g) 24 h and 96 h post-treatment with SARS-CoV-2 spike

Fig. 6

Prolonged expression of genes associated with proinflammatory pathways mediating the vascular inflammatory effect of SARS-CoV-2 spike protein. A segment of the KEGG pathway showing DE genes associated with SARS-CoV-2 spike-activated HPMC at 24 h (a) and 96 h (b) were significantly enriched in the TNF signaling pathway

Fig. 7

Enrichment of antigen presentation and coagulation cascade at 96 h. KEGG pathway map depicting the enrichment of antigen processing and presentation (a) as well as the complement and coagulation pathway (b) in the spike-treated HPMC at 96 h