Figure 4.

Recombinant viral antigens S and N increase vascular permeability and death without endothelial activation. (A–D) Vascular organoids were treated with spike glycoprotein (S) (100 nM), nucleocapsid (N), or a combination of both proteins for 72 h in the presence or absence of IL1-β (20 ng/mL) compared to control was measured by flow cytometry. (A) Representative flow cytometry blots of EC treated with S, N, and S and N antigens in the presence and absence of IL1-β. (B) Fold change in the live population of CD31⁺ cells from the total population using Live/Dead fixable dead cell stain. (C) Fold change in CD144 expression on CD31 ⁺ cells compared to untreated organoids and (D) fold change in ICAM-1 expression on CD31⁺ cells in treated organoids vs. untreated were measured by flow cytometry. (E) Detection of the levels of soluble IL6, (F) IL8, and (G) VWF in the supernatant of organoids treated for 72 h with viral antigens in the presence or absence of IL-1β by ELISA (n = 3 independent experiments, 3–4 replicate per experiment). (H) Representative flow cytometry plots of pericytes treated with S, N, and S and N antigens in the presence and absence of IL1-β (20 ng/mL). (I) Live/dead NG2⁺ cells from the total population, (J) fold change in ICAM-1 expression on CD140 ⁺ NG2⁺ cells in treated organoids vs. untreated were measured by flow cytometry. (K) qPCR of vascular organoids treated with combination S and N proteins for 0, 4, 24, and 72 h for genes regulating endothelial permeability. N = 3 for experiments, where one replicate is a total of 12–15 individual organoids pooled for assays from each of three independent biological replicates (independent differentiations). One-way ANOVA with multiple comparisons performed for each statistical test with significance at (* P = <0.05, ** P = <0.01, *** P = <0.001, **** P = <0.0001).