Figure 10.

Pro-inflammatory effect of SARS-CoV-2 or Spike S1 protein on HUVECs. (A) RT-qPCR for gene expression analysis of MCP-1/CCL2, RANTES/CCL5, IP-10/CXCL10, IL-8/CXCL8, IL-6 and TGF-β in SARS-CoV-2-stimulated HUVECs. After 24 h of treatment with S1, total mRNA was isolated and gene expression analysis was performed by RT-qPCR. The expression was normalized using the housekeeping genes 18S, ACTB and GAPDH; the results were mediated and expressed as fold increase. Data are expressed as mean ± SD of two independent experiments performed in triplicate. *p < 0.05; **p < 0.01; ***p < 0.001, as compared to untreated cells. (B) Analysis of the expression of the adhesion molecules VCAM-1 and E-Selectin. Four different populations of HUVECs were grown to confluence in a 96-well plate and incubated with 10 ng/mL or 1000 ng/mL of Spike S1 protein. TNF-α (100 ng/mL) was used as a positive control. Cells were incubated with anti-human VCAM-1 or anti-human E-selectin monoclonal antibodies, followed by alkaline phosphatase-conjugated secondary antibodies. Data are expressed as mean ± SE of four experiments performed in triplicate. **p < 0.01, as compared to untreated cells (Mann-Whitney test). (C) Permeabilizing effect of Spike protein on endothelial cells. The permeabilizing activity was evaluated kinetically, after 15 and 30 minutes, by adding 10 ng/mL or 1000 ng/mL of Spike protein to the upper chamber of the transwell, and then measuring the amount of FITC-labeled BSA that leaked through a monolayer of endothelial cells into the lower chamber. Histamine (HIS) was used as a positive control. Data are expressed as mean ± SD of four experiments performed in duplicate. *p < 0.05; **p < 0.01, as compared to untreated cells (Mann-Whitney test).