Figure 4.

SARS-CoV-2 activates human ECs through p38 MAPK/NF-κB with and without ACE2

(A) Western blotting analysis at 72 h after viral exposure showing that p38 MAPK, NF-κB, and eNOS pathways were significantly elevated in both ACE2-deficient and -expressing hESC-ECs, respectively, compared with their respective mock infection controls; but the degrees of activation of p38 MAPK and NF-κB pathways were significantly attenuated by endothelial ACE2 expression. (B) p38 MAPK, (C) p-p38 MAPK at Thr180/Tyr182, (D) p-p38 MAPK/MAPK, (E) NF-κB, (F) p-NF-κB at Ser536, (G) p-NF-κB/NF-κB, (H) IL-1β, (I) eNOS, (J) p-eNOS at Ser632, and (K) p-eNOS/eNOS. Data are presented as mean ± SD, n = 6, the relative protein levels were compared with the mock infection controls (value = 1) of each time point, differences were determined by one-way ANOVA and Tukey’s HSD post hoc test, ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001. (L and M) Monocyte adhesion assays showing increased numbers of adhered fluorescence-labeled THP-1 (red) to hESC-ECs at 72 h after inoculation compared with mock infection controls that were significantly reduced by CLI095 or dexamethasone. DAPI (blue) indicates cell nuclei. Scale bar, 200 μm. Data are presented as mean ± SD, n = 8, differences were determined by one-way ANOVA and Tukey’s HSD post hoc test, ^∗p < 0.05, ^∗∗p < 0.01. (N) NO production assays showing significantly reduced NO probes detected in hESC-ECs treated with virus and CLI095 compared with the virus alone group or mock infection control group. Data are presented as mean ± SD, n = 4, the relative probe intensities were compared with that of the mock infection controls (value = 1), differences were determined by one-way ANOVA and Tukey’s HSD post hoc test, ^∗p < 0.05.