Figure 5.

Human ECs can recognize and be activated by SARS-CoV-2 through TLR4

(A–K) (A) Western blotting analysis at 72 h after viral exposure showing that p38 MAPK, NF-κB, and eNOS pathways were significantly increased in ACE2-deficient hESC-ECs and HUVEC compared with their respective mock infection controls; and activation of p38 MAPK and NF-κB pathways were aborted by TLR blockade through CLI095 or dexamethasone in HUVEC. (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. (B–K) Data are presented as mean ± SD, n = 3–6, the relative protein levels were compared with hESC-EC mock infection controls (value = 1), differences were determined by two-way ANOVA and Tukey’s HSD post hoc test, ^∗p < 0.05, ^∗∗p < 0.01 compared within the same cell type; or ^#p < 0.05, ^##p < 0.01, ^###p < 0.001 compared with the same treatment conditions of another cell type. (L and M) Monocyte adhesion assays showing significantly increased numbers of adhered fluorescence-labeled THP-1 (red) to HUVEC at 72 h after inoculation compared with mock infection controls that were significantly blocked 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. (N) NO production assays showing significantly increased NO probes detected in HUVEC at 72 h after inoculation compared with mock infection controls that were significantly aborted by CLI095. Data are presented as mean ± SD, n = 4, the relative probe intensities were compared with that of mock infection controls (value = 1), differences were determined by one-way ANOVA and Tukey’s HSD post hoc test, ^∗p < 0.05.